Precision medicine by targeting vegf-a variants for treatment of diabetic macular edema and branch retinal vein occlusion

ABSTRACT

The invention relates to human targets of interest (TOI), anti-TOI ligands, kits compositions and method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 14/331,730 filed Jul. 15, 2014, the entire content of which isincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 14, 2014, isnamed 069496-080095_SL.txt and is 222,931 bytes in size.

TECHNICAL FIELD

The technology described herein relates to ligands, e.g., antibodies forthe treatment of disease.

BACKGROUND

It is recognized that individual humans differ in their sequence andrecently several individuals have had their genomes sequenced, forinstance James Watson and Craig Venter. Comparison of the genomesequence of individuals has revealed differences in their sequences inboth coding and non-coding parts of the genome. Some of these variationsin humans are significant and contribute to phenotypic differencesbetween individuals. In extreme cases these will result in geneticdisease. The 1000 Genomes Project has the objective of cataloguingsequences in the human genome, involving sequencing the genomes of avery large sampling of individuals from diverse art-recognized humanethnic populations.

SUMMARY

Through the application of human genetic variation analysis andrationally-designed sequence selection the present invention providesfor improved human patient diagnosis and therapy. Importantly, theinvention enables tailored medicines that address individual humanpatient genotypes or phenotypes.

The inventor's analysis of large numbers of naturally-occurring genomichuman TOI sequences reveals that there is significant variation acrossdiverse human populations and provides for the ability for correlationbetween individual human patients and tailored medical and diagnosticapproaches addressing the target. The technical applications of thesefindings, as per the present invention, thus contribute to bettertreatment, prophylaxis and diagnosis in humans and provides for patientbenefit by enabling personalized medicines and therapies. This providesadvantages of better prescribing, less wastage of medications andimproved chances of drug efficacy and better diagnosis in patients.

Furthermore, the inventor surprisingly realised that some rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention and better servingpatients in those populations.

With this, the inventor realised that there is significant industrialand medical application for the invention in terms of guiding the choiceof an anti-TOI ligand for administration to human patients for therapyand/or prophylaxis of TOI-mediated or associated diseases andconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

To this end, the invention provides:—

In a First Configuration

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   a. Administering to the human an anti-TOI ligand to target a TOI    variant in the human and treat or prevent said disease or condition,    wherein the TOI in said human is encoded by a nucleotide sequence    having a cumulative human allele frequency of less than 50% and/or    wherein the TOI in said human is encoded by a nucleotide sequence    having a total human genotype frequency of less than 50%;

wherein

-   b. Before step (a) said human has been or is genotyped as positive    for said nucleotide sequence or phenotyped as positive for said TOI    variant.

In a Second Configuration

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is encoded by a        nucleotide sequence having a cumulative human allele frequency        of less than 50% and/or wherein the TOI in said human is encoded        by a nucleotide sequence having a total human genotype frequency        of less than 50%; wherein    -   b. before step (a) the ligand has been or is determined as        capable of binding to said TOI variant.

In a Third Configuration

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   a. Administering to the human an anti-TOI ligand to target a TOI    variant in the human and treat or prevent said disease or condition,    wherein the TOI in said human is a variant encoded by a nucleotide    sequence having a cumulative human allele frequency of more than 50%    and/or having a total human genotype frequency of more than 50%;    wherein-   b. Before step (a) said human has been or is genotyped as negative    for a variant nucleotide sequence having a cumulative human allele    frequency of less than 50% and/or having a total human genotype    frequency of less than 50%; or phenotyped as negative for a TOI    variant encoded by a nucleotide sequence having a cumulative human    allele frequency of less than 50% and/or having a total human    genotype frequency of less than 50%.

In a Fourth Configuration

An anti-human TOI ligand for use in a method of treating and/orpreventing a TOI-mediated disease or condition in a human, wherein theTOI is present in humans as different polymorphic variants and whereinthe genome of said human comprises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or a total humangenotype frequency of less than 50%, the method comprising administeringthe ligand to the human.

In a Fifth Configuration

A ligand that binds a human TOI comprising an amino acid sequenceencoded by a TOI nucleotide sequence having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50%, for use in a method comprising the step of using theligand to target said TOI in a human to treat and/or prevent a diseaseor condition mediated by TOI, the method comprising administering theligand to the human.

In a Sixth Configuration

A pharmaceutical composition or kit for treating or preventing acondition or disease mediated by a TOI.

In a Seventh Configuration

A method of producing an anti-human TOI antibody binding site, themethod comprising obtaining a plurality of anti-TOI antibody bindingsites, screening the antibody binding sites for binding to a TOIcomprising an amino acid sequence encoded by a nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, or to a peptide thereofthat comprises an amino acid variation from the corresponding sequenceencoded by the TOI-encoding nucleotide sequence having the highestcumulative human allele frequency and/or the highest total humangenotype frequency, and isolating an antibody binding site that binds inthe screening step.

In a Eighth Configuration

A method of producing an anti-human TOI antibody, the method comprisingimmunising a non-human vertebrate (eg, a mouse or a rat) with a TOIcomprising an amino acid sequence encoded by a nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, or to a peptide thereofthat comprises an amino acid variation from the corresponding sequenceencoded by the TOI-encoding nucleotide sequence having the highestcumulative human allele frequency and/or the highest total humangenotype frequency, and isolating an antibody that binds a TOIcomprising an amino acid sequence encoded by a TOI nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, and optionallyproducing a TOI-binding fragment or derivative of the isolated antibody.

In a Ninth Configuration

A kit for TOI genotyping a human, wherein the kit comprises a nucleicacid comprising a nucleotide sequence that specifically hybridises to aTOI nucleotide sequence selected having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50% or an RNA transcript thereof; and/or the nucleic acidcomprises a nucleotide sequence that comprises at least 10 contiguousnucleotides of a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or a total human genotypefrequency of less than 50% or is an antisense sequence thereof.

In a Tenth Configuration

Use of an anti-TOI ligand that binds a human TOI comprising an aminoacid sequence encoded by a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, in the manufacture of a medicament fortreating and/or preventing a TOI-mediated disease or condition in ahuman whose genome comprises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or having a totalhuman genotype frequency of less than 50%.

In a Eleventh Configuration

Use of an anti-TOI ligand that binds a human TOI comprising an aminoacid sequence encoded by a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, in the manufacture of a medicament fortargeting said TOI in a human to treat and/or prevent a disease orcondition mediated by TOI.

In a Twelfth Configuration

A method of targeting a TOI for treating and/or preventing aTOI-mediated disease or condition in a human, the method comprisingadministering an anti-TOI ligand to a human comprising a TOI nucleotidesequence selected having a cumulative human allele frequency of lessthan 50% and/or a total human genotype frequency of less than 50%,whereby a TOI encoded by said nucleotide sequence is targeted.

In a Thirteenth Configuration

A method of TOI genotyping a nucleic acid sample of a human, the methodcomprising identifying in the sample the presence of a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or having a total human genotype frequency of less than 50%.

In a Fourteenth Configuration

A method of TOI typing a protein sample of a human, the methodcomprising identifying in the sample the presence of a TOI amino acidsequence encoded by a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or having a total human genotypefrequency of less than 50%.

In an example, the TOI is a human TOI selected from the group consistingof PCSK9, VEGF-A and IL6 receptor.

A Fifteenth Configuration provides a ligand, method, use, kit orcomposition of the invention, wherein

(i) the ligand (eg, antibody or fragment) comprises

-   -   (a) a variable domain that is encoded by a human V region        nucleotide sequence, wherein the V nucleotide sequence is        derived from recombination of human VH, D and JH gene segments        or human VL and JL gene segments; or    -   (b) a constant region domain encoded by a C region gene segment;    -   Wherein a first gene segment of said gene segments of (a), or        said C region gene segment of (b) comprises a first single        nucleotide polymorphism (SNP) encoding a first amino acid        polymorphism; and        (ii) the genome of said human comprises said first SNP or        wherein said human expresses (a′) an antibody variable domain        comprising said first amino acid polymorphism or (b′) an        antibody constant domain comprising said first amino acid        polymorphism.

A Sixteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand comprises or consistsof an antibody or fragment that comprises a human antibody variabledomain derived from the recombination of a human V gene segment and ahuman J gene segment (and optionally a human D gene segment when thevariable domains are VH domains); and wherein the genome of the humancomprises said human V gene segment and/or the human expressesantibodies comprising antibody variable domains derived from therecombination of said human V gene segment and a human J gene segment(and optionally a human D gene segment).

A Sixteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused human PCSK9receptor) comprises a human heavy chain constant domain encoded by afirst constant region nucleotide sequence; and wherein the genome of thehuman comprises a heavy chain constant region nucleotide sequence thatis identical to said first constant region nucleotide sequence and/orthe human expresses antibodies comprising said human constant domain.

A Seventeenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused TOI receptor)comprises a human gamma-1 heavy chain constant region that comprises anAsp corresponding to position 204 of SEQ ID NO: 42 or a Leucorresponding to position 206 of SEQ ID NO: 42 and wherein the genome ofthe human comprises a gamma-1 heavy chain constant region nucleotidesequence that encodes such an Asp or Leu or the human expressesantibodies comprising human gamma-1 constant regions comprising such anAsp or Leu.

A Eighteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused TOI receptor)comprises a human gamma-2 heavy chain constant region that comprises anamino acid selected from the group consisting of a Pro corresponding toposition 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Valcorresponding to position 161 of SEQ ID NO: 44 and an Ala correspondingto position 257 of SEQ ID NO: 44; and wherein the genome of the humancomprises a gamma-2 heavy chain constant region nucleotide sequence thatencodes such a selected amino acid or the human expresses antibodiescomprising human gamma-2 constant regions comprising such a selectedamino acid.

A Ninteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused TOI receptor)comprises a human kappa light chain constant region that comprises a Valcorresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding toposition 87 of SEQ ID NO: 50; and wherein the genome of the humancomprises a kappa light chain constant region nucleotide sequence thatencodes such a Val or Cys or the human expresses antibodies comprisinghuman kappa light chain constant regions comprising such a Val or Cys.

A Twentieth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand comprises or consistsof an antibody or fragment, wherein the antibody or fragment comprises aVH domain that is derived from the recombination of a human VH genesegment, a human D gene segment and a human JH gene segment, wherein theVH gene segment is selected from the group consisting of (i) IGHV1-18*01and the genome of the human comprises a human IGHV1-18*01 nucleotidesequence or the human expresses antibodies comprising variable domainsderived from the recombination of human IGHV1-18*01; or (ii) IGVH1-46*01and the genome of the human comprises a human IGHV1-46*01 nucleotidesequence or the human expresses antibodies comprising variable domainsderived from the recombination of human IGHV146*01.

A Twenty-First Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand comprises or consistsof an antibody or fragment, wherein the antibody or fragment comprises aVL domain that is derived from the recombination of a human VL genesegment and a human JL gene segment, wherein the VL gene segment isselected from the group consisting of (i) IGKV4-1*01 and the genome ofthe human comprises a human IGKV4-1*01 nucleotide sequence or the humanexpresses antibodies comprising variable domains derived from therecombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome ofthe human comprises a human IGLV2-14*01 nucleotide sequence or the humanexpresses antibodies comprising variable domains derived from therecombination of human IGLV2-14*01; or (iii) IGKV1-13*02 and the genomeof the human comprises a human IGKV1-13*02 nucleotide sequence or thehuman expresses antibodies comprising variable domains derived from therecombination of human IGKV1-13*02.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows in silico modeling of PCSK9 surface variant residues.

FIG. 2 depicts the cumulative allele frequency distribution across the1000 Genomes Project databse of human VH3-23 alleles comprising SNPrs56069819 (such alleles denonted “C” and the most frequent allele(which does not comprise this SNP) denoted “A”). The figure shows thatVH3-23 alleles comprising SNP rs56069819 are present at a cumulativefrequency of 11% across all human ethnic populations taken as a whole,whereas in certain specific human ethnic sub-populations (ASW, LWK, YRI,CEU and GBR) such alleles are present at an above-average cumulativefrequency. Indicated in the figure are those human PCSK9 variant forms(marked “Variants”) that are found in the various sub-populations withabove-average occurrence of human VH3-23 alleles comprising SNPrs56069819.

FIG. 3 depicts frameworks and CDRs encoded by VH3-23*04 as obtained fromthe IMGT database (available on the World Wide Web at www.IMGT.org).FIG. 3 discloses the nucleotide sequences as SEQ ID NOS 68, 68, 68, 70,70, 70, 71, 73, 75, 39 and 76, respectively, in order of appearance.FIG. 3 discloses the coded amino acid sequences as SEQ ID NOS 69, 69,69, 69, 69, 69, 72, 74, 74, 38 and 77, respectively, in order ofappearance.

FIG. 4 depicts sequences of VH3-23*04. The portion of VH3-23*04comprising the FW1 residue change of rs56069819 (SEQ ID NO: 38). Theportion of the nucleic acid sequence encoding rs56069819 is depicted(SEQ ID NO: 39). The FW1 encoded by VH3-23*04 is depicted (SEQ ID NO:40).

DETAILED DESCRIPTION

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in conformation oractivity of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to tailor medicines anddiagnosis of patients more effectively. The present invention providesfor tailored pharmaceuticals and testing that specifically addressesrarer variant forms of a human target of interest (TOI).

The present invention harnesses the power of human genetic variationanalysis and rationally-designed sequence selection. The technicalapplications of these approaches, as per the present invention,contribute to better treatment, prophylaxis and diagnosis in humans andprovides for patient benefit by providing choice and enablingpersonalized medicines and therapies. This provides advantages of betterprescribing, less wastage of medications and improved chances of drugefficacy and better diagnosis in patients.

As sources of genomic sequence variation data, the skilled person willbe aware of the available databases and resources (including updatesthereof) provided by the following:—

-   1. HapMap (The International HapMap Consortium. 2003; available on    the World Wide Web at hapmap.ncbi.nlm.nih.gov/index.html.en). The    HapMap Project is an international project that aims to compare the    genetic sequences of different individuals to identify chromosomal    regions containing shared genetic variants. The HapMap www site    provides tools to identify chromosomal regions and the variant    therein, with options to drill down to population level frequency    data.-   2. 1000 Genomes Project (The 1000 Genomes Project Consortium 2010;    available on the World Wide Web at 1000genomes.org/). This resource    provides complete genomic sequence for at least 2500 unidentified    individuals from one of 25 distinct population groups.-   3. Japanese SNP Database (H. Haga et al. 2002; available on the    World Wide Web at snp.ims.u-tokyo.ac.jp/index.html). Based on a    study identifying 190,562 human genetic variants.

The present invention involves the identification and cataloguing ofnaturally-occurring human genomic target sequence variants, includingthose found to be relatively low-frequency or rare variants thatsegregate with specific human ethnic populations and in many individualhumans.

An aspect of the invention is based on rational design of sequenceselection addressing the desirability to tailor medicaments anddiagnostics to rarer, but yet still significant groups of humanindividuals that suffer from, or have the potential to suffer from (ie,who are at risk of), a disease or condition mediated or associated withthe target of interest. In devising this rational design of the presentaspect of the invention, the inventor included considerations of thespread of prevalence of naturally-occurring target variant sequencesacross multiple, diverse human ethnic populations, as well as theimportance of addressing such populations where many individuals arelikely to display a genotype and/or phenotype of one or more of thevariants being analysed. As part of this design, the inventor saw theimportance of adopting the art-recognised classifications of humanethnic populations, and in this respect the inventor based the analysisand design on the recognised human ethnic populations adopted by the1000 Genomes Project, since this is a resource that is, and willcontinue to be, widely adopted by the scientific and medical community.

Thus, in this aspect of the invention, the inventor designed thefollowing variant sequence selection criteria, these being criteria thatthe inventor realised would provide for useful medical drugs anddiagnostics to tailored need in the human population.

Selection Criteria

Three or four of the following:—

-   -   Naturally-occurring human target variant sequences having a        cumulative human allele frequency of 35% or less;    -   Naturally-occurring human target variant sequences having a        total human genotype frequency of 40% or less;    -   Naturally-occurring human target variant sequences found in many        different human ethnic populations (using the standard        categorisation of the 1000 Genomes Project; see Table 4 below);        and    -   Naturally-occurring human target variant sequences found in many        individuals distributed across such many different ethnic        populations.

The inventor's selection included, as a consideration, selection fornucleotide variation that produced amino acid variation in correspondingTOI forms (ie, non-synonymous variations), as opposed to silentvariations that do not alter amino acid residues in the target protein.

In an embodiment, the cumulative human allele frequency is 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about5%.

In an embodiment, the naturally-occurring human target variant sequencesare found in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20different human ethnic populations (using the standard categorisation ofthe 1000 Genomes Project).

In an embodiment, the naturally-occurring human target variant sequencesare found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individualsdistributed across such many different ethnic populations.

In an example, the following criteria are applied:—

-   -   Naturally-occurring human target variant sequences having a        cumulative human allele frequency of 15% or less;    -   Naturally-occurring human target variant sequences having a        total human genotype frequency of 20% or less;    -   Naturally-occurring human target variant sequences found in at        least 5 different human ethnic populations (using the standard        categorisation of the 1000 Genomes Project); and    -   Naturally-occurring human target variant sequences found in many        individuals distributed across such many different ethnic        populations.

In an example, the criteria are applied with reference to one or morehuman genomic sequence databases as described herein. For example, thecriteria are those as applied to the 1000 Genomes database.

For example in any aspect example, embodiment or configuration of theinvention, the 1000 Genomes database release 13. For example, the 1000Genomes database in its most recent version as at 1 Oct. 2013.

Optionally, further sequence analysis and 3D in silico modelling (eg,see FIG. 1) can also be used as an additional selection criterion:variants whose variant amino acid residues (versus the most common formof human TOI) are surface-exposed on the target are desirable forselection, since the inventor saw these as contributing to determiningthe topography of the target and potentially contributing to how andwhere ligand binding on the target occurs.

The following bioinformatics protocol is envisaged to identify humansequences for use in the present invention:

-   -   (a) Identify a genomic region containing a target sequence of        interest (target genomic region′) and calculate the genomic        coordinates, using coordinates that match the sequence assembly        build used by either the 1000 Genomes Project or International        HapMap project (or another selected human gene database of        choice).    -   (b) Identify genomic variants mapped to the genomic region        previously identified in (a). Retrieve allele frequencies for        variants for each super population and preferably sub-population        where such data is available. The VWC tools for the 1000 Genomes        Project can be used for this step.    -   (c) Filter list of genomic variants from target genomic region        to contain only variants classed as either ‘non-synonymous’        single nucleotide polymorphisms (SNPs) or genomic ‘insertions or        delections’ (indels). Filter further to include those that are        present in exonic sequences only. “Non-synonymous” refers to        nucleotide variation that produces amino acid variation (ie,        excluding silent mutations).    -   (d) Correlate population frequency data for each of the        identified variants for each of the super populations (for        example ‘European Ancestry’, ‘East Asian ancestry’, ‘West        African ancestry’, ‘Americas’, and ‘South Asian ancestry’) to        identify those variants that segregate with less than two        super-populations. Further correlate all identified variants        with each of the sub-populations (for example, ‘European        ancestry’ super-population might be subdivided into groups such        as ‘CEU—Utah residents with Northern or Western European        ancestry’, ‘TSI Toscani in Italia’ and ‘British from England and        Scotland’) and produce a second score for rarity of variants        within a super-population.    -   (e) Collect one or more sequences that show segregation to        specific sub-populations for use in the present invention, eg,        according to selection criteria as described herein.

Human Populations

Optionally the ethnic populations are selected from those identified inthe 1000 Genomes Project database. In this respect, see Table 4 whichprovides details of the ethnic populations on which the 1000 GenomesProject database is based.

N A Rosenberg et al (Science 20 Dec. 2002: vol. 298 no. 5602 2342-2343)studied the genetic structure of human populations of differinggeographical ancestry. In total, 52 populations were sampled, thesebeing populations with:

African Ancestry

(Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers ofNiger-Kordofanian languages (Bantu, Yoruba or Mandenka populations),

Eurasian Ancestry

(European ancestry (Orcadian, Adygel, Basque, French, Russians,Italians, Sardinian, Tuscan),

Middle Eastern ancestry (Mozabite, Bedouin, Druze, Palestinians),

Central/South Asian ancestry (Balochl, Brahul, Makrani, Sindhi, Pathan,Burusho, Hazara, Uygur, Kalash)),

East Asian Ancestry

(Han, Dal, Daur, Hezhen, Lahu, Miao, Orogen, She, Tujia, Tu, Xibo, Yi,Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ancestry(Melanesian, Papuan); or

Americas Ancestry

(Karitiana, Surui, Colombian, Maya, Pima).

The International HapMap Project, Nature, 2003 Dec. 18;426(6968):789-96, discloses that goal of the HapMap Project: todetermine the common patterns of DNA sequence variation in the humangenome by determining the genotypes of one million or more sequencevariants, their frequencies and the degree of association between themin DNA samples from populations with ancestry from parts of Africa, Asiaand Europe. The relevant human populations of differing geographicalancestry include Yoruba, Japanese, Chinese, Northern European andWestern European populations. More specifically:—

Utah population with Northern or Western European ancestry (samplescollected in 1980 by the Centre d'Etude du Polymorphisme Humain (CEPH));

population with ancestry of Yoruba people from Ibadan, Nigeria;population with Japanese ancestry; andpopulation with ancestry of Han Chinese from China.

The authors, citing earlier publications, suggest that ancestralgeography is a reasonable basis for sampling human populations.

A suitable sample of human populations used in the present invention isas follows:—

-   -   (a) European ancestry    -   (b) Northern European ancestry; Western European ancestry;        Toscani ancestry; British ancestry, Finnish ancestry or Iberian        ancestry.    -   (c) More specifically, population of Utah residents with        Northern and/or Western European ancestry; Toscani population in        Italia; British population in England and/or Scotland; Finnish        population in Finland; or Iberian population in Spain.    -   (a) East Asian ancestry    -   (b) Japanese ancestry; Chinese ancestry or Vietnamese ancestry.    -   (c) More specifically, Japanese population in Tokyo, Japan; Han        Chinese population in Beijing, China; Chinese Dai population in        Xishuangbanna; Kinh population in Ho Chi Minh City, Vietnam; or        Chinese population in Denver, Colo., USA.    -   (a) West African ancestry    -   (b) Yoruba ancestry; Luhya ancestry; Gambian ancestry; or        Malawian ancestry.    -   (c) More specifically, Yoruba population in Ibadan, Nigeria;        Luhya population in Webuye, Kenya; Gambian population in Western        Division, The Gambia; or Malawian population in Blantyre,        Malawi.    -   (a) Population of The Americas    -   (b) Native American ancestry; Afro-Caribbean ancestry; Mexican        ancestry; Puerto Rican ancestry; Columbian ancestry; or Peruvian        ancestry.    -   (c) More specifically, population of African Ancestry in        Southwest US; population of African American in Jackson, Miss.;        population of African Caribbean in Barbados; population of        Mexican Ancestry in Los Angeles, Calif.; population of Puerto        Rican in Puerto Rico; population of Colombian in Medellin,        Colombia; or population of Peruvian in Lima, Peru.    -   (a) South Asian ancestry    -   (b) Ahom ancestry; Kayadtha ancestry; Reddy ancestry; Maratha;        or Punjabi ancestry.    -   (c) More specifically, Ahom population in the State of Assam,        India; Kayadtha population in Calcutta, India; Reddy population        in Hyderabad, India; Maratha population in Bombay, India; or        Punjabi population in Lahore, Pakistan.

In any configuration of the invention, in one embodiment, each humanpopulation is selected from a population marked “(a)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(b)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(c)” above.

In one embodiment the ethnic populations are selected from the groupconsisting of an ethnic population with European ancestry, an ethnicpopulation with East Asian, an ethnic population with West Africanancestry, an ethnic population with Americas ancestry and an ethnicpopulation with South Asian ancestry.

In one embodiment the ethnic populations are selected from the groupconsisting of an ethnic population with Northern European ancestry; oran ethnic population with Western European ancestry; or an ethnicpopulation with Toscani ancestry; or an ethnic population with Britishancestry; or an ethnic population with Icelandic ancestry; or an ethnicpopulation with Finnish ancestry; or an ethnic population with Iberianancestry; or an ethnic population with Japanese ancestry; or an ethnicpopulation with Chinese ancestry; or an ethnic population Vietnameseancestry; or an ethnic population with Yoruba ancestry; or an ethnicpopulation with Luhya ancestry; or an ethnic population with Gambianancestry; or an ethnic population with Malawian ancestry; or an ethnicpopulation with Native American ancestry; or an ethnic population withAfro-Caribbean ancestry; or an ethnic population with Mexican ancestry;or an ethnic population with Puerto Rican ancestry; or an ethnicpopulation with Columbian ancestry; or an ethnic population withPeruvian ancestry; or an ethnic population with Ahom ancestry; or anethnic population with Kayadtha ancestry; or an ethnic population withReddy ancestry; or an ethnic population with Maratha; or an ethnicpopulation with Punjabi ancestry.

Anti-Target Ligands

The invention provides useful anti-target ligands for addressing humanssuffering from or likely to suffer from a disease or condition mediatedor associated with the TOI. For example, the ligand specifically bindsto the TOI variant as per the invention. The ligand may inhibit orantagonise the activity of the target, eg, the ligand neutralises thetarget. The skilled person will be familiar with neutralising ligands ingeneral, such as antibodies or antibody fragments, and can readily testsuitable ligands for specific binding and/or neutralisation of a targetin vitro or in an in vivo assay.

An antibody “fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include dAb, Fab, Fab′, F(ab′)2and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

In an embodiment, the ligand of the invention is or comprises anantibody or antibody fragment, for example an antibody or fragmentcomprising human variable regions (and optionally also human constantregions). Anti-TOI or TOI-binding or targeting antibodies and fragmentscan be prepared according to any known method, eg, using transgenic mice(eg, the Kymouse™ or Velocimouse™, or Omnimouse™, Xenomouse™, HuMabMouse™ or MeMo Mouse™), rats (eg, the Omnirat™), camelids, sharks,rabbits, chickens or other non-human animals immunised with the TOIfollowed optionally by humanisation of the constant regions and/orvariable regions to produce human or humanised antibodies. In anexample, display technologies can be used, such as yeast, phage orribosome display, as will be apparent to the skilled person. Standardaffinity maturation, eg, using a display technology, can be performed ina further step after isolation of an antibody lead from a transgenicanimal, phage display library or other library. Representative examplesof suitable technologies are described in US20120093818 (Amgen, Inc),which is incorporated herein by reference, eg, the methods set out inparagraphs [0309] to [0346]. Although this is with reference to PCSK9,the antibody-generating methods can be applied to other TOIs as per thebroadest scopes of the present invention.

Generally, a VELOCIMMUNE™ or other mouse or rat can be challenged withthe antigen of interest, and lymphatic cells (such as B-cells) arerecovered from the mice that express antibodies. The lymphatic cells maybe fused with a myeloma cell line to prepare immortal hybridoma celllines, and such hybridoma cell lines are screened and selected toidentify hybridoma cell lines that produce antibodies specific to theantigen of interest. DNA encoding the variable regions of the heavychain and light chain may be isolated and linked to desirable isotypicconstant regions of the heavy chain and light chain. Such an antibodyprotein may be produced in a cell, such as a CHO cell. Alternatively,DNA encoding the antigen-specific chimaeric antibodies or the variabledomains of the light and heavy chains may be isolated directly fromantigen-specific lymphocytes.

Initially, high affinity chimaeric antibodies are isolated having ahuman variable region and a mouse constant region. As described below,the antibodies are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Themouse constant regions are replaced with a desired human constant regionto generate the fully human antibody of the invention, for examplewild-type or modified IgG1 or IgG4 (for example, SEQ ID NO: 751, 752,753in US2011/0065902, which sequences are incorporated herein by referencefor use in the ligands of the present invention). While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

In an example, the ligand of the invention is or comprises a nucleicacid, eg, RNA, eg, siRNA that hybridises under stringent condition tothe TOI variant sequence, eg, hybridises a nucleotide sequencecomprising one or more nucleotides that are variant (versus the mostcommon TOI sequence, eg, with reference to the 1000 Genomes Projectdatabase).

Target binding ability, specificity and affinity (Kd, K_(off) and/orK_(on)) can be determined by any routine method in the art, eg, bysurface plasmon resonance (SPR). The term “Kd”, as used herein, isintended to refer to the equilibrium dissociation constant of aparticular antibody-antigen interaction.

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0.05% by volume, eg, in the presence of P20 (polysorbate20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the ligand (eg, antibody) is determinedusing SPR by

-   1. Coupling anti-mouse (or other relevant human, rat or non-human    vertebrate antibody constant region species-matched) IgG (eg,    Biacore™ BR-1008-38) to a biosensor chip (eg, GLM chip) such as by    primary amine coupling;-   2. Exposing the anti-mouse IgG (or other matched species antibody)    to a test IgG antibody to capture test antibody on the chip;-   3. Passing the test antigen over the chip's capture surface at 1024    nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and-   4. And determining the affinity of binding of test antibody to test    antigen using surface plasmon resonance, eg, under an SPR condition    discussed above (eg, at 25° C. in physiological buffer). SPR can be    carried out using any standard SPR apparatus, such as by Biacore™ or    using the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, eg, using amodel inherent to the ProteOn XPR36™ analysis software.

In an example, the ligand of the invention is contained in a medicalcontainer, eg, a vial, syringe, IV container or an injection device (eg,an intraocular or intravitreal injection device). In an example, theligand is in vitro, eg, in a sterile container. In an example, theinvention provides a kit comprising the ligand of the invention,packaging and instructions for use in treating or preventing ordiagnosing in a human a disease or condition mediated by the TOI. In anexample, the instructions indicate that the human should be genotypedfor a TOI variant sequence of the invention before administering theligand to the human. In an example, the instructions indicate that thehuman should be phenotyped for a TOI variant of the invention beforeadministering the ligand to the human. In an example, the human is ofChinese (eg, Han) ethnicity and the instructions are in Chinese (eg,Mandarin). In an example, the instructions comprise directions toadminister alirocumab or evolocumab to said human.

The invention relates to the concepts set out in the following clauses.

Clause 1

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent (eg, by at least 40,        50, 60, 70, 80, 90 or 95%) said disease or condition, wherein        the TOI in said human is encoded by a nucleotide sequence having        a cumulative human allele frequency of less than 50% and/or        wherein the TOI in said human is encoded by a nucleotide        sequence having a total human genotype frequency of less than        50%;        -   wherein        -   Before step (a) said human has been or is genotyped as            positive for said nucleotide sequence or phenotyped as            positive for said TOI variant, or the method comprises            before step (a) genotyping the human as positive for said            nucleotide sequence or phenotyping the human as positive for            said TOI variant.

In any aspect, configuration, example, embodiment, clause or conceptherein, frequencies may be determined using bioinformatics.

In any aspect, configuration, example, embodiment, clause or conceptherein, frequencies may be determined by reference to a databasecomprising at least 1000 or 2000 human sequences.

In any aspect, configuration, example, embodiment, clause or conceptherein “heterozygous human genotype frequency” means the cumulativefrequency of all genotypes in the sample or database or in humans havingone occurrence of the rare variant allele and one occurrence of anotherallele (heterozygous state), eg, genotype in 1000 Genomes database.

In any aspect, configuration, example, embodiment, clause or conceptherein “homozygous human genotype frequency means the cumulativefrequency of two occurrences of the variant allele (homozygous state),eg, genotype in 1000 Genomes Project database.

In any aspect, configuration, example, embodiment, clause or conceptherein “total human genotype frequency” means the total of heterozygousplus homozygous human genotype frequencies.

In any aspect, configuration, example, embodiment, clause or conceptherein “cumulative human allele frequency” refers to the total of alloccurrences of the variant allele in the sample or database or inhumans, eg, in the 1000 Genomes Project database.

Clause 2:

The method of clause 1, wherein before step (a) the ligand has been oris determined as being capable of binding to said TOI variant, eg, withan affinity (Kd) disclosed below.

In an example, the ligand is (or has been determined as) a neutraliserof the TOI. In an example, determination is carried out in a human (eg,in a clinical trial). In an example, determination is carried out in anon-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-humanprimate (eg, Cynomolgous monkey, rhesus monkey or baboon).

Clause 3:

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent (eg, by at least 40,        50, 60, 70, 80, 90 or 95%) said disease or condition, wherein        the TOI in said human is encoded by a nucleotide sequence having        a cumulative human allele frequency of less than 50% and/or        wherein the TOI in said human is encoded by a nucleotide        sequence having a total human genotype frequency of less than        50%;        -   wherein    -   b. Before step (a) the ligand has been or is determined as        capable of binding to said TOI variant, eg, with an affinity        (Kd) disclosed below.

In an example, the ligand is (or has been determined as) a neutraliserof the TOI. In an example, determination is carried out in a human (eg,in a clinical trial). In an example, determination is carried out in anon-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-humanprimate (eg, Cynomolgous monkey, rhesus monkey or baboon).

Clause 4:

The method of clause 3, wherein the genome of said human comprises anucleotide sequence encoding said TOI variant; and before step (a) saidnucleotide sequence has been or is determined as having a cumulativehuman allele frequency of less than 50% and/or having a total humangenotype frequency of less than 50%.

The TOI variant is not the most frequent.

Clause 5:

The method of clause 3 or 4, wherein said human has been or is genotypedas positive for said variant nucleotide sequence before step (a), or themethod comprises genotyping the human as positive for said variantnucleotide sequence before step (a).

Clause 6:

The method of any preceding clause, wherein the human has been or isphenotyped as positive for said TOI variant before step (a), or themethod comprises phenotyping the human as positive for said variantnucleotide sequence before step (a).

Clause 7:

The method of any preceding clause, wherein said frequency is less than10 or 15% (eg, from 1 to 10%).

In an embodiment, the cumulative human allele frequency is 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about5%.

Clause 8:

The method of any preceding clause, wherein the ligand is capable ofbinding to two or more different TOI variants, each being encoded by anucleotide sequence having a cumulative human allele frequency of lessthan 50% (eg, from 1 to 10%) and/or having a total human genotypefrequency of less than 50% (eg, from 1 to 20%).

In an embodiment, the cumulative human allele frequency of each TOIvariant is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to20% or 1 to 15% or 1 to 10%.

In an embodiment, the total human genotype frequency of each TOI variantis 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%,1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5%or 1 to about 5%.

Clause 9:

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent (eg, by at least 40,        50, 60, 70, 80, 90 or 95%) said disease or condition, wherein        the TOI in said human is a variant encoded by a nucleotide        sequence having a cumulative human allele frequency of more than        50% (eg, the highest frequency) and/or having a total human        genotype frequency of more than 50% (eg, the highest frequency);        -   wherein    -   b. Before step (a) said human has been or is genotyped as        negative for a variant nucleotide sequence having a cumulative        human allele frequency of less than 50% and/or having a total        human genotype frequency of less than 50%; or phenotyped as        negative for a TOI variant encoded by a nucleotide sequence        having a cumulative human allele frequency of less than 50%        and/or having a total human genotype frequency of less than 50%;        -   or        -   Before step (a) said the method comprises genotyping the            human as negative for a variant nucleotide sequence having a            cumulative human allele frequency of less than 50% and/or            having a total human genotype frequency of less than 50%; or            phenotyping the human as negative for a TOI variant encoded            by a nucleotide sequence having a cumulative human allele            frequency of less than 50% and/or having a total human            genotype frequency of less than 50%.

In an embodiment, in (a) the cumulative human allele frequency is 55,60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or100% (eg, in the range from 51 to 80%).

In an embodiment, in (a) the total human genotype frequency is 55, 60,65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or100% (eg, in the range from 51 to 80%).

In an embodiment, in (b) the cumulative human allele frequency is 30,25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15%or 1 to 10%.

In an embodiment, in (b) the total human genotype frequency is 35, 30,25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%,1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 toabout 5%.

Clause 10:

The method of clause 9, wherein before step (a), the human has been oris phenotyped as positive for the most frequent TOI variant or genotypedfor the nucleotide sequence thereof

In an embodiment, before step (a) the human has been or is genotyped aspositive for TOI variant nucleotide sequence having a cumulative humanallele frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but lessthan 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%) orphenotyped for the TOI variant thereof

In an embodiment, before step (a) the human has been or is genotyped aspositive for TOI variant nucleotide sequence having a total humangenotype frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but lessthan 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%) orphenotyped for the TOI variant thereof

Clause 11:

The method of clause 9 or 10, wherein before step (a) the ligand hasbeen or is determined as being capable of binding to the most frequentTOI variant.

Clause 12:

The method of clause 9, 10 or 11, wherein before step (a) the ligand hasbeen or is determined as being substantially incapable of neutralisingor inhibiting said TOI variant recited in step (b).

By “substantially incapable or neutralising or inhibiting” is meant:Neutralisation or inhibition less than 50, 25, 10, 5 or 0.5% inhibitionor neutralisation of the most frequent TOI variant.

Clause 13:

The method of any one of clauses 9 to 12, wherein the ligand is capableof binding to the most frequent TOI variant.

Clause 14:

The method of any one of clauses 9 to 13, wherein the ligand is capableof binding to two or more different TOI variants, each being encoded bya nucleotide sequence having a cumulative human allele frequency of morethan 50%.

In an embodiment, each TOI variant is encoded by a nucleotide sequencehaving a cumulative human allele frequency of 55, 60, 65, 70, 75, 80, 85or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the rangefrom 51 to 80%).

In an embodiment, each TOI variant is encoded by a nucleotide sequencehaving a total human genotype frequency of 55, 60, 65, 70, 75, 80, 85 or90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the rangefrom 51 to 80%).

Clause 15:

The method of any preceding clause, wherein said variant nucleotidesequence recited in step (a) has been or is determined as being presentin at least 2 different human ethnic populations, eg, at least 2, 3, 4,5, 6, 7, 8 or 9 different human ethnic populations in Table 4.

Clause 16:

The method of any preceding clause, wherein said human frequency is thefrequency in a database of naturally-occurring sequences sampled from atleast 15, 20 or 25 different human ethnic populations and comprising atleast 1000 sequences. In an embodiment, the database is the 1000 GenomesProject database as described herein.

Clause 17:

An anti-human TOI ligand for use in a method of treating and/orpreventing a TOI-mediated disease or condition in a human, wherein theTOI is present in humans as different polymorphic variants and whereinthe genome of said human comprises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or a total humangenotype frequency of less than 50%, the method comprising administeringthe ligand to the human.

In the alternative, clause 17 provides an anti-human TOI ligand for usein a method according to any one of clauses 1 to 16, the methodcomprising administering the ligand to the human.

In an embodiment, the cumulative human allele frequency is 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about5%.

Clause 18:

The ligand of clause 17, wherein the ligand has been or is determined ascapable of binding the human TOI encoded by said nucleotide sequence.

In the alternative, clause 18 provides a ligand that binds a human TOIcomprising an amino acid sequence encoded by a TOI nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, for use in a methodcomprising the step of using the ligand to target said TOI in a human totreat and/or prevent a disease or condition mediated by TOI, the methodcomprising administering the ligand to the human.

Clause 19:

A ligand that binds a human TOI comprising an amino acid sequenceencoded by a TOI nucleotide sequence having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50%, for use in a method according to any one of clauses 1 to16, the method comprising administering the ligand to the human.

Clause 20:

The ligand of any one of clauses 17 to 19, wherein the human has been oris genotyped as positive for said TOI nucleotide sequence having acumulative human allele frequency of less than 50%.

The ligand of any one of clauses 17 to 19, wherein the human has been oris genotyped as positive for said TOI nucleotide sequence having a totalhuman genotype frequency of less than 50%.

Clause 21:

The ligand of any one of clauses 17 to 20, wherein the human has been oris phenotyped as positive for a TOI encoded by a nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/orhaving a total human genotype frequency of less than 50%.

Clause 22:

The ligand of any one of clauses 17 to 21, wherein the human has been oris genotyped as heterozygous for a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or having a totalhuman genotype frequency of less than 50%; optionally wherein the humanhas been or is genotyped as comprising a TOI nucleotide sequence havinga cumulative human allele frequency of less than 50% and a TOInucleotide sequence having a cumulative human allele frequency of morethan 50% (eg, having the highest cumulative human allele frequency)and/or having a total human genotype frequency of more than 50% (eg,having the highest total human genotype frequency).

Clause 23:

The ligand of any one of clauses 17 to 22, wherein the genome of thehuman has been or is genotyped as homozygous for a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or having a total human genotype frequency of less than 50%.

Clause 24:

The ligand of any one of clauses 17 to 23, wherein the ligand comprisesan antibody binding site that binds a human TOI comprising an amino acidsequence encoded by a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or having a total human genotypefrequency of less than 50%; and optionally has been or is determined ascapable of such binding.

Clause 25:

The ligand of clause 24, wherein the ligand is an antibody or antibodyfragment.

Clause 26:

The ligand of any one of clauses 17 to 23, wherein the ligand comprisesa nucleotide sequence that specifically hybridises to a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or having a total human genotype frequency of less than 50% or anRNA transcript thereof; and/or the ligand comprises a nucleotidesequence that comprises at least 10 contiguous nucleotides of anucleotide sequence having a cumulative human allele frequency of lessthan 50% and/or having a total human genotype frequency of less than 50%or is an antisense sequence thereof

In an embodiment, the ligand comprises a nucleotide sequence thatcomprises at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or100 contiguous nucleotides of a nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or having a total humangenotype frequency of less than 50% or is an antisense sequence thereof

Clause 27:

The ligand of any one of clauses 17 to 26, wherein the genome of saidhuman comprises a nucleotide sequence having a cumulative human allelefrequency of less than 50% and the sequence is found in at least 2different ethnic populations (eg, found in at least 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 different human ethnic populations (forexample as per the populations in Table 4)). In an example, numbers arewith reference to the 1000 Genomes Project database.

The ligand of any one of clauses 17 to 26, wherein the genome of saidhuman comprises a nucleotide sequence having a cumulative human allelefrequency of less than 50% and the sequence is found in at least 20individuals distributed across at least 2 said different ethnicpopulations (eg, found in at least in at least 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130,140 or 150 individuals distributed across such many different ethnicpopulations). In an example, numbers are with reference to the 1000Genomes Project database.

Clause 28:

A pharmaceutical composition or kit for treating or preventing acondition or disease mediated by a TOI as recited in any precedingclause, the composition or kit comprising a ligand of any one of clauses17 to 27; and optionally in combination with a label or instructions foruse to treat and/or prevent said disease or condition in a human;optionally wherein the label or instructions comprise a marketingauthorisation number (eg, an FDA or EMA authorisation number);optionally wherein the kit comprises an injection pen or IV containerthat comprises the ligand.

In an example, the label or instructions cover or describe use for ahuman comprising a TOI variant encoded by a nucleotide sequence asrecited in clause 17.

Clause 29:

A method of producing an anti-human TOI antibody binding site, themethod comprising obtaining a plurality of anti-TOI antibody bindingsites, screening the antibody binding sites for binding to a TOIcomprising an amino acid sequence encoded by a nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, or to a peptide thereofthat comprises an amino acid variation from the corresponding sequenceencoded by the TOI-encoding nucleotide sequence having the highestcumulative human allele frequency and/or the highest total humangenotype frequency, and isolating an antibody binding site that binds inthe screening step.

In an embodiment of any aspect herein, the antibody, fragment or bindingsite is recombinant

In the alternative, clause 29 provides: A method of producing ananti-human TOI antibody, the method comprising immunising a non-humanvertebrate (eg, a mouse or a rat) with a TOI comprising an amino acidsequence encoded by a nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, or to a peptide thereof that comprises anamino acid variation from the corresponding sequence encoded by theTOI-encoding nucleotide sequence having the highest cumulative humanallele frequency and/or the highest total human genotype frequency, andisolating an antibody that binds a TOI comprising an amino acid sequenceencoded by a TOI nucleotide sequence having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50%, and optionally producing a TOI-binding fragment orderivative of the isolated antibody.

The term “isolated” with reference to a ligand, antibody or protein, forexample in any aspect, configuration, example or embodiment, means thata subject ligand, antibody, protein etc (1) is free of at least someother proteins with which it would normally be found, (2) is essentiallyfree of other proteins from the same source, e.g., from the samespecies, (3) is expressed by a cell from a different species, (4) hasbeen separated from at least about 50 percent of polynucleotides,lipids, carbohydrates, or other materials with which it is associated innature, (5) is operably associated (by covalent or noncovalentinteraction) with a polypeptide with which it is not associated innature, or (6) does not occur in nature. Typically, an “isolated”ligand, antibody, protein etc constitutes at least about 5%, at leastabout 10%, at least about 25%, or at least about 50% of a given sample.Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or anycombination thereof can encode such an isolated ligand, antibody proteinetc. Preferably, the isolated ligand, antibody protein etc issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

For example, an “isolated” antibody is one that has been identified,separated and/or recovered from a component of its productionenvironment (eg, naturally or recombinantly). Preferably, the isolatedpolypeptide is free of association with all other components from itsproduction environment, eg, so that the antibody has been isolated to anFDA-approvable or approved standard. Contaminant components of itsproduction environment, such as that resulting from recombinanttransfected cells, are materials that would typically interfere withresearch, diagnostic or therapeutic uses for the antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide will be purified: (1)to greater than 95% by weight of antibody as determined by, for example,the Lowry method, and in some embodiments, to greater than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, an isolated polypeptide or antibodywill be prepared by at least one purification step.

Immunoconjugates

The invention encompasses the ligand (eg, antibody) conjugated to atherapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxinagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxin agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, see for example, WO 05/103081.

Bispecifics

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific mAbs may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al. (1991) J. Immunol. 147:60-69. The human anti-TOI (eg, anti-PCSK9)mAbs can be linked to or co-expressed with another functional molecule,e.g., another peptide or protein. For example, an antibody or fragmentthereof can be functionally linked (e.g., by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody or antibody fragment, toproduce a bispecific or a multispecific antibody with a second bindingspecificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)CH3 domain and a second Ig CH3 domain, wherein the first and second IgCH3 domains differ from one another by at least one amino acid, andwherein at least one amino acid difference reduces binding of thebispecific antibody to Protein A as compared to a bi-specific antibodylacking the amino acid difference. In one embodiment, the first Ig CH3domain binds Protein A and the second Ig CH3 domain contains a mutationthat reduces or abolishes Protein A binding such as an H95R modification(by IMGT exon numbering; H435R by EU numbering). The second CH3 mayfurther comprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, andV82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the presentinvention.

Clause 30:

The method of clause 29, comprising the step of obtaining a nucleic acidencoding the antibody, fragment, derivative or binding site andoptionally inserting the nucleic acid in an expression vector.

Clause 31:

A kit for TOI genotyping a human, wherein the kit comprises a nucleicacid comprising a nucleotide sequence that specifically hybridises to aTOI nucleotide sequence selected having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50% or an RNA transcript thereof and/or the nucleic acidcomprises a nucleotide sequence that comprises at least 10 (eg, at least10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100) contiguousnucleotides of a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or a total human genotypefrequency of less than 50% or is an antisense sequence thereof

For example, the nucleic acid hybridises to a region immediatelyflanking a nucleotide that is variant compared to the correspondingnucleotide of the TOI nucleotide sequence having the highest cumulativehuman allele frequency and/or the highest total human genotypefrequency. In an example, the nucleic acid hybridises to at two or moresuch variant nucleotides.

Specific hybridisation is under stringent conditions, as will beapparent to the skilled person, eg, conditions of 5×SSC, 5×Denhardt'sreagent, and 0.5% SDS at 65° C.

Clause 32:

A kit for TOI genotyping or phenotyping a human, wherein the kitcomprises a ligand according to any one of clauses 17 to 27 or anantibody, fragment or derivative produced by the method of any one ofclauses 29 to 31.

For example, the ligand specifically binds to an epitope comprising anamino acid that is variant compared to the corresponding amino acid ofthe TOI encoded by a nucleotide sequence having the highest cumulativehuman allele frequency and/or the highest total human genotypefrequency. In an example, the ligand specifically binds to an epitopecomprising two or more such variant amino acids. In an example, specificbinding means binding with an affinity (Kd) of 1 mM, 100 nM, 10 nM or 1nM or less, eg, as determined by SPR.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

Clause 33:

Use of an anti-TOI ligand that binds a human TOI comprising an aminoacid sequence encoded by a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, in the manufacture of a medicament fortreating and/or preventing a TOI-mediated disease or condition in ahuman whose genome comprises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or having a totalhuman genotype frequency of less than 50%.

Clause 34:

Use of an anti-TOI ligand that binds a human TOI comprising an aminoacid sequence encoded by a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, in the manufacture of a medicament fortargeting said TOI in a human to treat and/or prevent a disease orcondition mediated by TOI.

The use of clause 33 or 34, wherein the ligand, human, disease orcondition is according to any one of clauses 1 to 27.

Clause 35:

A method of targeting a TOI for treating and/or preventing aTOI-mediated disease or condition in a human, the method comprisingadministering an anti-TOI ligand to a human comprising a TOI nucleotidesequence selected having a cumulative human allele frequency of lessthan 50% and/or a total human genotype frequency of less than 50%,whereby a TOI encoded by said nucleotide sequence is targeted.

Clause 36:

The method of clause 35, wherein the method comprises targeting a humanTOI comprising an amino acid sequence with said ligand to treat and/orprevent said disease or condition in said human, wherein said amino acidsequence is encoded by a nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or a total human genotypefrequency of less than 50%.

Clause 37:

A method of TOI genotyping a nucleic acid sample of a human, the methodcomprising identifying in the sample the presence of a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or having a total human genotype frequency of less than 50%.

In an example, the method comprises obtaining a TOI nucleic acid samplefrom the human and then carrying out the identifying step.

Clause 38:

A method of TOI typing a protein sample of a human, the methodcomprising identifying in the sample the presence of a TOI amino acidsequence encoded by a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or having a total human genotypefrequency of less than 50%.

In an example, the method comprises obtaining a TOI protein sample fromthe human and then carrying out the identifying step.

Clause 39:

The method of clause 37 or 38, comprising obtaining a sample of serum,blood, faeces, hair, urine or saliva from a human, whereby the nucleicacid or protein sample is obtained for use in the step of identifyingsaid sequence.

Clause 40:

The method of any one of clauses 37 to 39, comprising using a ligandaccording to any one of clauses 17 to 27 to carry out said identifyingstep.

Clause 41:

A diagnostic kit comprising a ligand that is capable of binding a humanTOI comprising an amino acid sequence encoded by a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or a total human genotype frequency of less than 50% andinstructions for carrying out the method of clause 38 or 39.

Clause 42:

A diagnostic kit comprising a nucleic acid probe comprising a nucleotidesequence that specifically hybridises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or a total humangenotype frequency of less than 50% or an RNA transcript thereof andinstructions for carrying out the method of clause 38 or 39.

Clause 43:

The method, ligand, composition, kit or use of any preceding clause,wherein the TOI is encoded by a nucleotide sequence having a cumulativehuman allele frequency from 1 to 10% and/or a total human genotypefrequency from 1 to about 15% or from 1 to 15%.

Clause 44:

The method, ligand, composition, kit or use of any preceding clausewherein the TOI is a human TOI selected from Table 5; optionally fortreating and/or preventing a corresponding disease or condition as setout in Table 5.

For example, the TOI is human PCSK9, eg, a mature, cleaved,autocatalysed or active PCSK9. In an example, the disease is acardiovascular disease such as hyperlipidaemia.

Ligands of the invention are useful, for instance, in specific bindingassays, for genotyping or phenotyping humans, affinity purification ofthe TOI and in screening assays to identify other antagonists of TOIactivity. Some of the ligands of the invention are useful for inhibitingbinding of TOI to a congnate human receptor or protein, or inhibitingTOI-mediated activities.

The invention encompasses anti-TOI (eg, PCSK9) antibody ligands having amodified glycosylation pattern. In some applications, modification toremove undesirable glycosylation sites may be useful, or e.g., removalof a fucose moiety to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In an example, the invention features a pharmaceutical compositioncomprising a ligand of the invention, wherein the ligand is or comprisesa recombinant human antibody or fragment thereof which specificallybinds the TOI (eg, a rare variant as described herein) and apharmaceutically acceptable carrier. In one embodiment, the inventionfeatures a composition which is a combination of an antibody ligand orantigen-binding fragment of an antibody of the invention, and a secondtherapeutic agent. The second therapeutic agent may be any of ananti-inflammatory agent, an anti-angiogenesis agent, a painkiller, adiuretic, a chemotherapeutic agent, an anti-neoplastic agent, avasodilator, a vasoconstrictor, a statin, a beta blocker, a nutrient, anadjuvant, an anti-obesity agent and an anti-diabetes agent.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the USA Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A “pharmaceutically acceptable carrier,excipient, or adjuvant” refers to an carrier, excipient, or adjuvantthat can be administered to a subject, together with an agent, e.g., anyantibody or antibody chain described herein, and which does not destroythe pharmacological activity thereof and is nontoxic when administeredin doses sufficient to deliver a therapeutic amount of the agent.

In an example, the invention features a method for inhibiting TOIactivity using the anti-TOI ligand of the invention (eg, an antibody orantigen-binding portion of the antibody of the invention), wherein thetherapeutic method comprises administering a therapeutically effectiveamount of a pharmaceutical composition comprising the ligand. Thedisorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by removal, inhibition or reductionof TOI activity.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

The term “specifically binds,” or the like, means that a ligand, eg, anantibody or antigen-binding fragment thereof, forms a complex with anantigen that is relatively stable under physiologic conditions. Specificbinding can be characterized by an equilibrium dissociation constant ofat least about 1×10⁻⁶M or less (e.g., a smaller KD denotes a tighterbinding). Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds a human TOI may, however, exhibitcross-reactivity to other antigens such as a TOI molecule from anotherspecies. Moreover, multi-specific antibodies (e.g., bispecifics) thatbind to human TOI and one or more additional antigens are nonethelessconsidered antibodies that “specifically bind” TOI, as used herein.

Genotyping & Phenotyping

The skilled person will be familiar with techniques that can be used foraccurate genotyping and application to the invention. These include thefollowing.

1 Hybridization-based methods1.1 Dynamic allele-specific hybridization1.2 Molecular beacons1.3 SNP microarrays2 Enzyme-based methods2.1 Restriction fragment length polymorphism2.2 PCR-based methods2.3 Flap endonuclease2.4 Primer extension2.5 5′-nuclease

2.6 Oligonucleotide Ligation Assay

3 Other post-amplification methods based on physical properties of DNA3.1 Single strand conformation polymorphism3.2 Temperature gradient gel electrophoresis3.3 Denaturing high performance liquid chromatography3.4 High-resolution melting of the entire amplicon3.5 Use of DNA mismatch-binding proteins3.6 SNPlex (SNPlex™ is a proprietary genotyping platform sold by AppliedBiosystems).

Next-generation sequencing technologies such as pyrosequencing is alsouseful.

Reference is also made to GB2444410A and the genotyping method disclosedtherein, which is incorporated herein by reference in its entirety.

Miniaturized assays, such as microarrays with oligonucleotide reagentsimmobilized on small surfaces, are frequently proposed for large-scalemutation analysis and high-throughput genotyping (Large-scaleidentification, mapping, and genotyping of single-nucleotidepolymorphisms in the human genome (Wang D G, Fan J B, Siao C J, Berno A,Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J,Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E,Mittmann M, Morris M S, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S,Hudson T J, Lipshutz R, Chee M, Lander E S, Science. 1998 May 15;280(5366):1077-82). Other high-throughput methods discriminate allelesby differential hybridization, primer extension, ligation and cleavageof an allele-specific probe (Review Accessing genetic variation:genotyping single nucleotide polymorphisms, Syvänen AC, Nat Rev Genet.2001 December; 2(12):930-42; Review Techniques patents for SNPgenotyping, Twyman R M, Primrose S B, Pharmacogenomics. 2003 January;4(1):67-79).

An approach for a fully automated, large-scale SNP analysis is the‘homogeneous’ assay, i.e. a single-phase assay without separation steps,permitting continual monitoring during amplification. The TaqMan™ assay(Applied Biosystems), originally designed for quantitative real-timePCR, is a homogeneous, single-step assay also used in determination ofmutation status of DNA (see, eg, A. A. Komar (ed.), Single NucleotidePolymorphisms, Methods in Molecular Biology 578, DOI10.1007/978-1-60327-411-1_19, Humana Press, a part of SpringerScience+Business Media, LLC; and Single Nucleotide Polymorphisms,Methods in Molecular Biology™ Volume 578, 2009, pp 293-306, The TaqManMethod for SNP Genotyping, Gong-Qing Shen et al). The TaqMan SNPGenotyping Assay exploits the 5′-exonuclease activity of AmpliTaq Gold™DNA polymerase to cleave a doubly labeled probe hybridized to theSNP-containing sequence of ssDNA. Cleavage separates a 5′-fluorophorefrom a 3′-quencher leading to detectable fluorescent signal. The use oftwo allele-specific probes carrying different fluorophores permits SNPdetermination in the same tube without any post-PCR processing. Genotypeis determined from the ratio of intensities of the two fluorescentprobes at the end of amplification. Thus, rather than taking advantageof the full set of real-time PCR data as in quantitative studies, onlyend-point data are used.

TaqMan SNP genotyping in a high-throughput, automated manner isfacilitated by the use of validated Pre-made TaqMan® Genotyping assays,but Custom TaqMan® Assays may also be used (High-throughput genotypingwith single nucleotide polymorphisms, Ranade K, Chang M S, Ting C T, PeiD, Hsiao C F, Olivier M, Pesich R, Hebert J, Chen Y D, Dzau V J, Curb D,Olshen R, Risch N, Cox D R, Botstein D, Genome Res. 2001 July;11(7):1262-8; Assessment of two flexible and compatible SNP genotypingplatforms: TaqMan SNP Genotyping Assays and the SNPlex GenotypingSystem, De la Vega F M, Lazaruk K D, Rhodes M D, Wenz M H, Mutat Res.2005 Jun. 3; 573(1-2):111-35). The results of the assay can beautomatically determined by genotyping software provided with real-timethermal cyclers (e.g. IQ software of Bio-Rad, Sequence DetectionSoftware of Applied Biosystems).

Single nucleotide polymorphisms (SNPs) can be determined using TaqManreal-time PCR assays (Applied Biosystems) and commercial software thatassigns genotypes based on reporter probe signals at the end ofamplification. An algorithm for automatic genotype caling of SNPs usingthe full course of TaqMan real-time data is available for use (A.Callegaro et al, Nucleic Acids Res. 2006; 34(7): e56, Published online2006 Apr. 14. doi: 10.1093/nar/gkl185, PMCID: PMC1440877). The algorithmis unique in that it classifies samples according to the behavior ofblanks (no DNA samples), which cluster with heterozygous samples. Thismethod of classification eliminates the need for positive controls andpermits accurate genotyping even in the absence of a genotype class, forexample when one allele is rare.

The skilled person will be familiar with techniques that can be used foraccurate phenotyping and application to the invention. These include theuse of amino acid sequencing of isolated target protein and comparisonof sequences from different variants (eg, with the most common variant).An antibody that specifically and selectively binds in the area of a SNPunder stringent conditions can also be used to identify a particularvariant. In another method, the genotype is determined and acorresponding amino acid sequence (phenotype) determined, eg, by insilico translation.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-TOIligand, eg, antibodies or antigen-binding fragments thereof, of thepresent invention. The administration of therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the ligand, eg, antibody, of the present invention isused for treating various conditions and diseases associated with theTOI in an adult patient, it is advantageous to intravenously administerthe antibody of the present invention normally at a single dose of about0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer theligand or pharmaceutical composition of the invention, for example aligand provided by e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the mutantviruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J.Biol. Chem. 262:4429-4432). Methods of introduction include, but are notlimited to, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The ligand orcomposition may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

The ligand or pharmaceutical composition can be also delivered in avesicle, in particular a liposome (see Langer (1990) Science249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss,New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.).

In certain situations, the ligand or pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng.14:201). In another embodiment, polymeric materials can be used; see,Medical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974). In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138, 1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a ligand or pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPENT™, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the ligand(s). Such dosage forms in a unit doseinclude, for example, tablets, pills, capsules, injections (ampoules),suppositories, etc. The amount of the aforesaid antibody contained isgenerally about 5 to about 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the aforesaidantibody is contained in about 5 to about 100 mg and in about 10 toabout 250 mg for the other dosage forms.

Exemplary TOIs

For example in any configuration, aspect, concept, example orconfiguration of the invention, the or each TOI is selected from thegroup consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1;ADORA2A; Aggrecan; AGR2; AICDA; AW1; AIG1; AKAP1; AKAP2; AIYIH;amyloid-beta; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC;APOC1; AR; Ax1; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF;BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); B1yS; BMP1; BMP2;BMP3B (GDF1O); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin);BRCA1; C19orflO (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1;CB1; CCBP2 (D6/JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13 (MCP-4);CCL15 (MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19(MIP-3b); CCL2 (MCP-1); MCAF; CCL2O (MIP-3a); CCL21 (MIP-2); SLC;exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 I eotaxin-2);CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3(MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2);CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA);CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD7,CD164; CD19; CD1C; CD2O; CD200; CD-22; CD24; CD28; CD3; CD37; CD38;CD3E; CD3G; CD3Z; CD4; CD4O; CD4OL; CD44; CD45RB; CD52; CD69; CD72;CD74; CD79A; CD79B; CD8; CD8O; CD81; CD83; CD86; CD96; CD207; CDH1(E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH2O; CDH5; CDH7;CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A(p21Wap1/Cip1); CDKN1B (p27Kip1); CDKNIC; CDKN2A (p161NK4a); CDKN2B;CDKN2C; CDKN3; CEBPB; CELSR3; CER1; CHGA; CHGB; Chitinase; CHRNG;CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8;CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1);CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CRLF2;CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsinB); CX3CL1 (SCYDi); CX3CR1 (V28); CXCR6; CXCL1 (GRO1); CXCL1O (IP-10);CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2(GRO2); CXCL3 (GRO3); CXCL5 (ENA-78 I LIX); CXCL6 (GCP-2); CXCL9 (MIG);CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR ISTRL33 I Bonzo); CYB5; CYC1;CYSLTR1; DAB2IP; DAND5; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1;EDG1; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3;EphA4; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD;FasL; FASN; FCER1A; FCER2; FCGR3A; FCRL4; FGF; FGF1 (aFGF); FGF1O;FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2(bFGF); FGF2O; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6(HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON);FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1(FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; Galectin-3;GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GHR; GM-CSF; GNAS1; GNRH1; GPR2(CCR1O); GPR31; GPR44; GPR81 (FKSG8O); GPR87; GPR137C; GRCC10 (C10);GRP; GSN (Gelsolin); GSTP1; HAVCR1; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9;hepcidin; hemojuvelin; HGF; HIF1A; HIP1; histamine and histaminereceptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOS; 1D2;IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; TFNW1;IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA;IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; 1L13;IL13RA1; IL13RA2; 1L14; 1L15; IL15RA; IL16; 1L17; IL17B; IL17C; IL17R;1L18; IL18BP; IL18R1; IL18RAP; 1L19; IL1A; IL1B; IL1F1O; IL1F5; IL1F6;IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2;IL1RL1; IL1RL2 IL1RN; 1L2; 1L20; IL2ORA; IL21R; 1L22; 1L22R; 1L22RA2;1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L28B; 1L29; IL2RA; IL2RB; IL2RG;1L3; 1L30; IL3RA; 1L4; IL4R; 1L5; IL5RA; 1L6; IL6 receptor; IL6ST(glycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK;INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; 1TGA3; ITGA6 (a6integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN;K6HF; KAI1; KDR; MTLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13;KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19);KRT2A; KRTHB6 (hair-specific type II keratin); LAG3; LAMAS; LEP(leptin); LIGHT; Lingo-p75; Lingo-Troy; LPS; LRP5; LTA (TNF-b); LTB;LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun);MDK; MIB1; midkine; MIF; MIP-2; MK167 (Ki-67); MMP2; MMP9; MS4A1; MSMB;MT3 (metallothionectin-ifi); MTSS 1; MUC 1 (mucin); MYC; MYD88; NCK2;neurocan; Na_(v)1.7; Na_(v)1.8; NFKB 1; NFKB2; NGFB (NGF); NGFR;NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5;NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3;NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1;NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPG;OPRD1; OX40L; OX40; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PCSK9,PD-1, PD-L1; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan;PIAS2; Placental Growth Factor (PIGF); PIK3CG; PLAU (uPA); PLG; PLXDC1;PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA;PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13;RGS3; RNF110 (ZNF144); ROBO2; ROR1; S100A2; SCGB1D2 (lipophilin B);SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelialMonocyte-activating cytokine); SDF2; SERPINA1; SERPINIA3; SERPINB5(maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1;SLC43A1; SLIT2; SPP1; SPRR1B (Spri); ST6GAL1; STAB1; STATE; STEAP;STEAP2; TB4R2; TBX21; TCP1O; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2;TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1);THBS2; THBS4; THPO; TIE (Tie-i); TIM3; TMP3; tissue factor; TLR1O; TLR2;TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TMPRSS6; TNF; TNF-α; TNFAIP2(B94); TNFAIP3; TNFRSF1 1A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5;TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF1O (TRAIL); TNFSF1 1(TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L);TNFSF1 5 (VEGI); TNFSF1 8; TNFSF4 (OX40 ligand); TNFSF5 (CD4O ligand);TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD3O ligand); TNFSF9 (4-1BBligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53;TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TRAIL;TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGFA; VEGFB; VEGFC; versican; VHL C5;VLA-4; Wnt7A; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1);YY1; and ZFPM2.

In an example, the TOI is a human TOI selected from Table 5.

In an example, the TOI is OX40 ligand.

In an example, the TOI is OX40.

In an example, the TOI is PCSK9.

In an example, the TOI is IL6 receptor (IL-6R).

In an example, the TOI is LIGHT.

In an example, the TOI is VEGF-A.

In an example, the TOI is TNF alpha.

In an example, the TOI is PIGF.

In an example, the TOI is IGF1R.

In an example, the TOI is OPG.

In an example, the TOI is ICOS

In an example, the TOI is NGF.

In an example, the TOI is BMP6.

In an example, the TOI is ferroportin.

In an example, the TOI is TMPRSS6.

In an example, the TOI is hemojuvelin.

In an example, the TOI is VEGF receptor.

In an example, the TOI is PDGF receptor.

In an example, the TOI is stem cell factor receptor.

In an example, the TOI is hepcidin.

In an example, the TOI is IL-4 receptor alpha.

In an example, the TOI is sclerostin.

In an example, the TOI is IL-13 receptor.

In an example, the TOI is CD7.

In an example, the TOI is delta-like ligand-4 (D114).

In an example, the TOI is HGF.

In an example, the TOI is angiopoietin-2 (Ang2).

In an example, the TOI is GDF8.

In an example, the TOI is ERBB3.

In an example, the TOI is IL-17 receptor.

In an example, the TOI is CD40.

In an example, the TOI is CD40 ligand.

In an example, the TOI is EGFR.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, or “reduction” are all used herein tomean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” typically means adecrease by at least 10% as compared to a reference level (e.g. theabsence of a given treatment) and can include, for example, a decreaseby at least about 10%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more. As used herein, “reduction” doesnot encompass a complete reduction as compared to a reference level. Adecrease can be preferably down to a level accepted as within the rangeof normal for an individual without a given disorder. However, forexample, for the purposes of lowering or reducing cholesterol level, forexample, a reduction by about 5-10 points can be considered a “decrease”or “reduction.”

In certain aspects of all embodiments of the invention, the term“inhibition” is used. Inhibition refers and refers to decrease by atleast 10% as compared to a reference level (e.g. the absence of a giventreatment) and can include, for example, a decrease by at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or more including 100% inhibition as compared to areference level. “Complete inhibition” refers to a 100% inhibition ascompared to a reference level.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, an “increase” is a statistically significant increasein such level.

As used herein, the term “substantially” refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena. For the removal ofdoubt, “substantially” can refer to at least a 90% extent or degree of acharacteristic or property of interest, e.g. at least 90%, at least 92%,at least 95%, at least 98%, at least 99% or greater.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. In some embodiments, thesubject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,” “patient” and “subject” are used interchangeably herein.In some embodiments, the subject can be a non-human vertebrate, e.g. aprimate, a rodent, a mouse, a rat, a pig, a sheep, a zebrafish, a frog,etc.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of adisease or condition, e.g., a cardiovascular condition. A subject can bemale or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatmentor one or more complications related to such a condition, andoptionally, have already undergone treatment for the condition or theone or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingthe condition or one or more complications related to the condition. Forexample, a subject can be one who exhibits one or more risk factors forthe condition or one or more complications related to the condition or asubject who does not exhibit risk factors.

A “subject in need” or “human in need” of treatment for a particularcondition can be a subject having that condition, such as increasedcholesterol levels, diagnosed as having that condition, or at risk ofdeveloping that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids with natural aminoacids. When referring to “modified polypeptides” one refers topolypeptides that include modified amino acids (e.g., phosphorylated,glycated, glycosylated, etc.) and amino acid analogs, regardless of itssize or function. “Protein” and “polypeptide” are often used inreference to relatively large polypeptides, whereas the term “peptide”is often used in reference to small polypeptides, but usage of theseterms in the art overlaps. The terms “protein” and “polypeptide” areused interchangeably herein when referring to a gene product andfragments thereof. Thus, exemplary polypeptides or proteins include geneproducts, naturally occurring proteins with the specified sequence. Onecan also use peptide homologs, peptide orthologs, peptide paralogs,peptide fragments and other equivalents, variants, fragments, andanalogs of the peptides as these terms are understood by one of ordinaryskill in the art.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid. The nucleic acid canbe either single-stranded or double-stranded. A single-stranded nucleicacid can be one nucleic acid strand of a denatured double-stranded DNA.Alternatively, it can be a single-stranded nucleic acid not derived fromany double-stranded DNA. In one aspect, the nucleic acid can be DNA. Inanother aspect, the nucleic acid can be RNA. Suitable nucleic acidmolecules are DNA, including genomic DNA or cDNA. Other suitable nucleicacid molecules are RNA, including mRNA. In some aspects one can also useanalogs of nucleic acids.

As used herein, the term “nucleic acid probe” refers to an isolatedoligonucleotide molecule having a nucleic acid sequence which canhybridize to a target nucleic acid sequence, e.g. specifically hybridizeto the target sequence. In some embodiments, a nucleic acid probe canfurther comprise a detectable label. In some embodiments, a nucleic acidprobe can be attached to a solid surface. In some embodiments, a nucleicacid from is from about 5 nt to about 100 nt in length.

As used herein, the term “siRNA” refers to a nucleic acid that forms anRNA molecule comprising two individual strands of RNA which aresubstantially complementary to each other. Typically, the siRNA is atleast about 15-40 nucleotides in length (e.g., each complementarysequence of the double stranded siRNA is about 15-40 nucleotides inlength, and the double stranded siRNA is about 15-40 base pairs inlength, preferably about 19-25 base nucleotides, e.g., 19, 20, 21, 22,23, 24, or 25 nucleotides in length). In some embodiments, a siRNA canbe blunt-ended. In some embodiments, a siRNA can comprise a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the overhang on one strand is not dependenton the length of the overhang on the second strand. The siRNA moleculescan also comprise a 3′ hydroxyl group. In some embodiments, the siRNAcan comprise a 5′ phosphate group. A siRNA has the ability to reduce orinhibit expression of a gene or target RNA when the siRNA is present orexpressed in the same cell as the target gene, e.g. the target RNA.siRNA-dependent post-transcriptional silencing of gene expressioninvolves cutting the target RNA molecule at a site guided by the siRNA.

As used herein, “PCSK9” or “proprotein convertase subtilisin/kexin type9” refers to a serine protease involved in regulating the levels of thelow density lipoprotein receptor (LDLR) protein (Horton et al., 2007;Seidah and Prat, 2007). PCSK9 has been shown to directly interact withthe LDLR protein, be endocytosed along with the LDLR, andco-immunofluoresce with the LDLR throughout the endosomal pathway(Lagace et al., 2006). PCSK9 is a prohormone-proprotein convertase inthe subtilisin (S8) family of serine proteases (Seidah et al., 2003).The sequence of PCSK9 for a variety of species is known, e.g., humanPCSK9 (NCBI Gene ID No: 255738). Nucleotide and polypeptide sequencesfor a number of PCSK9 isoforms are provided herein, e.g., SEQ ID NOs:1-37.

PCSK9 exists as both a pro-form and a mature form. Autocatalysis of thePCSK9 proform occurs between Gln152 and Ser153 (VFAQ|SIP (SEQ ID NO:67)) (Naureckiene et al., 2003), and has been shown to be required forits secretion from cells (Seidah et al., 2003). The inactive form priorto this cleavage can be referred to herein as the “inactive”,“pro-form”, or “unprocessed” form of PCSK9. The C-terminal fragmentgenerated by the autocatalysis event can be referred to herein as the“mature,” “cleaved”, “processed” or “active” PCSK9. Examples of pro-formand mature PCSK9 isoforms are provided herein, see, e.g. SEQ ID NOs:1-27.

As used herein, the “catalytic domain” of PCSK9 refers to the portion ofa PCSK9 polypeptide corresponding to positions 153 to 449 of PCSK9, e.g.of SEQ ID NO: 1. As used herein, the “C-terminal domain” of PCSK9 refersto the portion of a PCSK9 polypeptide corresponding to positions 450-692of PCSK9, e.g., of SEQ ID NO: 1.

As used herein, a disease or condition “mediated by PCSK9” refers to adisease or condition which is caused by or characterized by a change inPCSK9, e.g. a change in expression level, a change in activity, and/orthe presence of a variant or mutation of PCSK9. Non-limiting examples ofsuch diseases or conditions can include, for example, a lipid disorder,hyperlipoproteinemia, hyperlipidemia; dyslipidemia;hypercholesterolemia, a heart attack, a stroke, coronary heart disease,atherosclerosis, peripheral vascular disease, claudication, type IIdiabetes, high blood pressure, and a cardiovascular disease orcondition. In an example, the disease or condition is an inflammatory orautoimmune disease or condition. Methods of identifying and/ordiagnosing such diseases and conditions are well known to medicalpractitioners of ordinary skill.

A subject at risk of having or developing a disease or conditionmediated by PCSK9 can be a subject exhibiting one or more signs orsymptoms of such a disease or condition or having one or more riskfactors for such a disease or condition, e.g. being overweight, havingelevated cholesterol level, comprising one or more genetic polymorphismsknown to predispose to the disease or condition, e.g., elevatedcholesterol level, such as having a mutation in the LDLR (encodinglow-density lipoprotein receptor) or APOB (encoding apolipoprotein B) orin the PCSK9 gene and/or having a family history of such a disease orcondition.

As used herein, “ligand” refers to a molecule which can bind, e.g.,specifically bind, to a second molecule or receptor. In someembodiments, a ligand can be, e.g., an antibody, antibody fragment,antibody portion, and/or affibody.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from the polypeptide or nucleic acid (eg, the most commonone in humans, eg, most frequent in a database as disclosed herein, suchas the 1000 Genomes Project database) by one or more amino acid ornucleic acid deletions, additions, yet retains one or more specificfunctions or biological activities of the naturally occurring molecule.Amino acid substitutions include alterations in which an amino acid isreplaced with a different naturally-occurring amino acid residue. Suchsubstitutions may be classified as “conservative”, in which case anamino acid residue contained in a polypeptide is replaced with anothernaturally occurring amino acid of similar character either in relationto polarity, side chain functionality or size. Such conservativesubstitutions are well known in the art. Substitutions encompassed bythe present invention may also be “non-conservative”, in which an aminoacid residue which is present in a peptide is substituted with an aminoacid having different properties, such as naturally-occurring amino acidfrom a different group (e.g., substituting a charged or hydrophobicamino; acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. In some embodiments amino acid substitutions areconservative. Also encompassed within the term variant when used withreference to a polynucleotide or polypeptide, refers to a polynucleotideor polypeptide that can vary in primary, secondary, or tertiarystructure, as compared to a reference polynucleotide or polypeptide,respectively (e.g., as compared to a wild-type polynucleotide orpolypeptide).

Variants of PCSK9 are provided elsewhere herein. Variants of PCSK9 caninclude the forms described herein as a, f, c, r, p, m, e h, aj, and q.Sequences of these variants are provided herein, see, e.g, SEQ IDNOs:1-27 and in Table 1, 2 or 6.

In some aspects, one can use “synthetic variants”, “recombinantvariants”, or “chemically modified” polynucleotide variants orpolypeptide variants isolated or generated using methods well known inthe art. “Modified variants” can include conservative ornon-conservative amino acid changes, as described below. Polynucleotidechanges can result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence. Some aspects use include insertion variants, deletion variantsor substituted variants with substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. The term “conservativesubstitution,” when describing a polypeptide, refers to a change in theamino acid composition of the polypeptide that does not substantiallyalter the polypeptide's activity. For example, a conservativesubstitution refers to substituting an amino acid residue for adifferent amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the blood brainbarrier (BBB)). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.MoI Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986); 205-218 and S. French and B. Robson, J. MoI. Evol. 19 (1983)171. Accordingly, one can select conservative amino acid substitutionssuitable for amino acids on the exterior of a protein or peptide (i.e.amino acids exposed to a solvent), for example, but not limited to, thefollowing substitutions can be used: substitution of Y with F, T with Sor K, P with A, E with D or Q, N with D or G, R with K, G with N or A, Twith S or K, D with N or E, I with L or V, F with Y, S with T or A, Rwith K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulphide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria. Antibodies can be humanized usingroutine technology.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody fragment” refers to a polypeptidethat includes at least one immunoglobulin variable domain orimmunoglobulin variable domain sequence and which specifically binds agiven antigen. An antibody fragment can comprise an antibody or apolypeptide comprising an antigen-binding domain of an antibody. In someembodiments, an antibody fragment can comprise a monoclonal antibody ora polypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody fragment” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J.Immunol. 1996; 26(3):629-39; which is incorporated by reference hereinin its entirety)) as well as complete antibodies. An antibody can havethe structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesand combinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2,and CDR3), and Framework Regions (FRs). VH refers to the variable domainof the heavy chain. VL refers to the variable domain of the light chain.According to the methods used in this invention, the amino acidpositions assigned to CDRs and FRs may be defined according to Kabat(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991)) or according to IMGTnomenclature.

D domain or region refers to the diversity domain or region of anantibody chain. J domain or region refers to the joining domain orregion of an antibody chain

An antibody “gene segment”, e.g. a VH gene segment, D gene segment, orJH gene segment refers to oligonucleotide having a nucleic acid sequencethat encodes that portion of an antibody, e.g. a VH gene segment is anoligonucleotide comprising a nucleic acid sequence that encodes apolypeptide VH domain.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, IMGT or Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality.

As used herein, the term “antibody binding site” refers to a polypeptideor domain that comprises one or more CDRs of an antibody and is capableof binding an antigen. For example, the polypeptide comprises a CDR3(eg, HCDR3). For example the polypeptide comprises CDRs 1 and 2 (eg,HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (eg,HCDRs1-3). In an example, the antibody binding site is provided by asingle variable domain (eg, a VH or VL domain). In another example, thebinding site comprises a VH/VL pair or two or more of such pairs.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. For example, in an diagnostic test the specific binding of aligand can distinguish between two variant PCSK9 proteins as describedherein. In some embodiments, specific binding can refer to an affinityof the first entity for the second target entity which is at least 10times, at least 50 times, at least 100 times, at least 500 times, atleast 1000 times or greater than the affinity for the third nontargetentity. In the context of oligonucleotide strands which interact viahybridization, specific binding can be “specific hybridization.”

Additionally, and as described herein, a recombinant human(ized)antibody can be further optimized to decrease potential immunogenicity,while maintaining functional activity, for therapy in humans. In thisregard, functional activity means a polypeptide capable of displayingone or more known functional activities associated with a recombinantantibody or antibody reagent thereof as described herein. Suchfunctional activities include, e.g. the ability to bind to a targetmolecule.

The term “immunizing” refers to the step or steps of administering oneor more antigens to an animal so that antibodies can be raised in theanimal Generally, immunizing comprises injecting the antigen or antigensinto the animal. Immunization can involve one or more administrations ofthe antigen or antigens. Suitable methods are prime-boost and RIMMSprocedures as known to the skilled person in the art.

As used herein, an “affibody” refers to a relatively small syntheticprotein molecule that has high binding affinity for a target protein(e.g. for PCSK9 or a variant thereof). Affibodies are composed of athree-helix bundle domain derived from the IgG-binding domain ofstaphylococcal protein A. The protein domain consists of a 58 amino acidsequence, with 13 randomized amino acids affording a range of affibodyvariants. Despite being significantly smaller than an antibody (anaffibody weighs about 6 kDa while an antibody commonly weighs about 150kDa), an affibody molecule works like an antibody since its binding siteis approximately equivalent in surface area to the binding site of anantibody.

As used herein, “VH3-23*04” refers to a human VH domain variantcomprising the polypeptide sequence of SEQ ID NO: 38. As opposed to thereference sequence, VH3-23*04 has a valine residue instead of a leucineresidue (see FIGS. 3 and 4; L24V, numbering including signal sequence;valine at position 5 shown in FIG. 4) as a result of the presence of thers56069819 SNP in the nucleic acid sequence encoding the VH domain. Asused herein, “rs56069819” refers to a mutation or variant in a VH genesegment from adenosine to cytosine (or thymine to guanine, dependingupon the strand of DNA which is being read), resulting in the VH domainencoding VH3-23*04. Rs56069819 is depicted in FIG. 4 and SEQ ID NO: 39,which demonstrate the T->G mutation (it is noted that the dbSNP entryfor RS5606819 depicts the other strand, which comprises the A->Cmutation). Further description of VH3-23*04 can be found, e.g., in USPatent Publication 2013/0071405; which is incorporated by referenceherein in its entirety.

As used herein, “determine” or “determining” refers to ascertaining,e.g., by a quantitative or qualitative analysis. As used herein, “hasbeen determined” can refer to ascertaining on the basis of previouslyobtained information or simultaneously obtained information.

In some aspects of all embodiments of the invention selecting caninclude automation such as a computer implemented software program thatupon input of the relevant data such as ethnicity or a panel of SNP datacan make the determination based on the instructions set forth herein.

As used herein, “assaying” refers to assessing, evaluating, quantifying,measuring, or characterizing an analyte, e.g., measuring the level of ananalyte in a sample, identifying an analyte, or detecting the presenceor absence of an analyte in a sample. In some embodiments, assayingrefers to detecting a presence or absence of the analyte of interest. Insome embodiments, assaying refers to quantifying an amount of ananalyte, e.g., providing a measure of concentration or degree of analyteabundance. In some embodiments, assaying refers to enumerating thenumber of molecules of analyte present in a sample and/or specimen,e.g., to determine an analyte copy number.

As used herein “multiplex” refers to the carrying out of a method orprocess simultaneously and in the same reaction vessel on two or more,typically three or more, different target sequences, e.g. on two or moreisoforms of PCSK9, or PCSK9 and an additional target. A multiplexanalysis typically includes analysis of 10-50, 10-100; 10-1000, 10-5000,10-10000 reactions in a multiplex format, such as a multiwall, an array,or a multichannel reaction,

Often the analysis or multiplex analysis is also automated usingrobotics and typically software executed by a computer and may include arobotic handling of samples, automatic or robotic selection of positiveor negative results, assaying for presence of absence of a target, suchas a nucleic acid polymorphism or a protein variant.

The term “biological sample” or “test sample” as used herein denotes asample taken or isolated from a biological organism, e.g., a sample froma subject. Exemplary biological samples include, but are not limited to,a biofluid sample; serum; plasma; urine; saliva; hair, epithelial cells,skin, a tumor biopsy and/or tissue sample etc. The term also includes amixture of the above-mentioned samples. The term “test sample” or“biological sample” also includes untreated or pretreated (orpre-processed) biological samples. For the analysis of nucleic acids,the biological sample should typically comprise at least one cellcomprising nucleic acids.

The test sample can be obtained by removing a sample of cells from asubject, but can also be accomplished by using previously isolated cells(e.g. isolated at a prior time point and isolated by the same or anotherperson). In addition, the test sample can be freshly collected or apreviously collected, refrigerated, frozen or otherwise preservedsample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. Exemplary methods for treating a testsample include, but are not limited to, centrifugation, filtration,sonication, homogenization, heating, freezing and thawing, andcombinations thereof. In some embodiments, the test sample can be afrozen test sample, e.g., a frozen tissue. The frozen sample can bethawed before employing methods, assays and systems described herein.After thawing, a frozen sample can be centrifuged before being subjectedto methods, assays and systems described herein. In some embodiments,the test sample is a clarified test sample, for example, bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments, a test sample can be a pre-processedtest sample, for example, supernatant or filtrate resulting from atreatment selected from the group consisting of centrifugation,filtration, thawing, purification, and any combinations thereof. In someembodiments, the test sample can be treated with a chemical and/orbiological reagent. Chemical and/or biological reagents can be employedto protect and/or maintain the stability of the sample, includingbiomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. The skilled artisan is well aware of methods and processesappropriate for pre-processing of biological samples required fordetermination of the level of an expression product as described herein.

As used herein, “genotyping” refers to a process of determining thespecific allelic composition of a cell and/or subject at one or moreposition within the genome, e.g. by determining the nucleic acidsequence at that position. Genotyping refers to a nucleic acid analysisand/or analysis at the nucleic acid level. As used herein, “phenotyping”refers a process of determining the identity and/or composition of anexpression product of a cell and/or subject, e.g. by determining thepolypeptide sequence of an expression product. Phenotyping refers to aprotein analysis and/or analysis at the protein level.

As used herein, the term “nucleic acid amplification” refers to theproduction of additional copies of a nucleic acid sequence and istypically carried out using polymerase chain reaction (PCR) or ligasechain reaction (LCR) technologies well known in the art (Dieffenbach, C.W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y.). Other methods for amplificationare also contemplated in aspects of the invention.

The term “allele-specific amplification” refers to a reaction (e.g., PCRreaction) in which at least one of the primers (e.g., allele-specificprimer) is chosen from a polymorphic area of gene (e.g., singlenucleotide polymorphism), with the polymorphism located at or near theprimer's 3′-end. A mismatched primer will not initiate amplification,whereas a matched primer will initiate amplification. The appearance ofan amplification product is indicative of the presence of thepolymorphism.

As used herein, “sequencing” refers to the determination of the exactorder of nucleotide bases in a strand of DNA (deoxyribonucleic acid) orRNA (ribonucleic acid) or the exact order of amino acids residues orpeptides in a protein. Nucleic acid sequencing can be done using Sangersequencing or next-generation high-throughput sequencing.

As used herein “next-generation sequencing” refers to oligonucleotidesequencing technologies that have the capacity to sequenceoligonucleotides at speeds above those possible with conventionalsequencing methods (e.g. Sanger sequencing), due to performing andreading out thousands to millions of sequencing reactions in parallel.Non-limiting examples of next-generation sequencing methods/platformsinclude Massively Parallel Signature Sequencing (Lynx Therapeutics); 454pyro-sequencing (454 Life Sciences/Roche Diagnostics); solid-phase,reversible dye-terminator sequencing (Solexa/Illumina): SOLiD technology(Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNAnanoball sequencing (Complete Genomics); and technologies available fromPacific Biosciences, Intelligen Bio-systems, Oxford NanoporeTechnologies, and Helicos Biosciences. Next-generation sequencingtechnologies and the constraints and design parameters of associatedsequencing primers are well known in the art (see, e.g. Shendure, etal., “Next-generation DNA sequencing,” Nature, 2008, vol. 26, No. 10,1135-1145; Mardis, “The impact of next-generation sequencing technologyon genetics,” Trends in Genetics, 2007, vol. 24, No. 3, pp. 133-141; Su,et al., “Next-generation sequencing and its applications in moleculardiagnostics” Expert Rev Mol Diagn, 2011, 11(3):333-43; Zhang et al.,“The impact of next-generation sequencing on genomics”, J GenetGenomics, 2011, 38(3):95-109; (Nyren, P. et al. Anal Biochem 208: 17175(1993); Bentley, D. R. Curr Opin Genet Dev 16:545-52 (2006); Strausberg,R. L., et al. Drug Disc Today 13:569-77 (2008); U.S. Pat. No. 7,282,337;U.S. Pat. No. 7,279,563; U.S. Pat. No. 7,226,720; U.S. Pat. No.7,220,549; U.S. Pat. No. 7,169,560; U.S. Pat. No. 6,818,395; U.S. Pat.No. 6,911,345; US Pub. Nos. 2006/0252077; 2007/0070349; and 20070070349;which are incorporated by reference herein in their entireties).

As used herein, “nucleic acid hybridization” refers to the pairing ofcomplementary RNA and DNA strands as well as the pairing ofcomplementary DNA single strands. In some embodiments, nucleic acidhybridization can refer to a method of determining a nucleic acidsequence and/or identity by hybridizing a nucleic acid sample with aprobe, e.g. Northern or Southern blot analysis or microarray analysis.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment). For treatment to beeffective a complete cure is not contemplated. The method can in certainaspects include cure as well.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

Multiple compositions can be administered separately or simultaneously.Separate administration refers to the two compositions beingadministered at different times, e.g. at least 10, 20, 30, or 10-60minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One canalso administer compositions at 24 hours apart, or even longer apart.Alternatively, two or more compositions can be administeredsimultaneously, e.g. less than 10 or less than 5 minutes apart.Compositions administered simultaneously can, in some aspects, beadministered as a mixture, with or without similar or different timerelease mechanism for each of the components.

As used herein, “contacting” refers to any suitable means fordelivering, or exposing, an agent to at least one complex, enzyme, orcell. Exemplary delivery methods include, but are not limited to, directdelivery to cell culture medium, perfusion, injection, or other deliverymethod well known to one skilled in the art.

As used herein, “obtain” refers to any method of acquiring, securing,procuring, or coming into the possession of, e.g. a sample. Obtaining abiological sample from a subject can comprise physical removing a samplefrom a subject (e.g. drawing blood or taking a hair or saliva sample)without or without active participation from the subject; receiving asample from a subject (e.g. the subject collects a saliva or hair samplethemselves and provides it, e.g. in a container provided for thepurpose); or procuring a sample from a storage facility, medicalfacility, or medical provider. Obtain from the human or subject, refersto an active step of, e.g., drawing blood or taking a tissue or cellsample.

As used herein, “cholesterol level” refers to a level of one or more oftotal cholesterol, LDL cholesterol, HDL cholesterol, and/ortriglycerides. Cholesterol levels can be the level of cholesterol in theblood of a subject.

As used herein in reference to cholesterol levels, “maintain” refers topreventing the level from worsening (e.g. increasing). In someembodiments, maintaining a particular level refers to a process thatresults in the cholesterol level not increasing by more than 10% overtime. Maintaining may also refer to maintaining a previously achievedlevel. For example, if a human has received statin treatment, one canmaintain the cholesterol level achieved using the statin treatment.

In some embodiments, the subject treated according to the methodsdescribed herein has previously had their cholesterol level reduced. Asused herein, “previously reduced” indicates that at a prior point intime, the subject experienced a decrease in cholesterol levels. Thedecrease can be due to administration of a pharmaceutical composition(e.g. administration of a composition as described herein or anothercomposition, e.g. a statin) or due to another cause, e.g. a change indiet and/or exercise.

An existing treatment for high cholesterol levels is the administrationof a statin. As referred to herein, a “statin” (also known as HMG-CoAreductase inhibitors) are inhibitors of the enzyme HMG-coA reductase,which mediates cholesterol production in the liver. Statins, bycompetitively binding HMG-CoA reductase, prevent the binding of HMG-CoAto the enzyme and thereby inhibit the activity of the reductase (e.g.the production of mevalonate). Non-limiting examples of statins caninclude atorvastatin (LIPITOR™), fluvastatin (LESCOL™), lovastatin(MEVACOR™, ALTOCOR™), pitavastatin (LIVALO™), pravastatin (PRAVACHOL™)rosuvastatin (CRESTOR™), and simvastatin (ZOCOR™). Statins can beadministered in combination with other agents, e.g. the combination ofezetimibe and simvastatin.

Some subjects are, or become, resistant to statin treatment. As usedherein, “resistant to statin treatment” or “reduced responsiveness tostatin treatment” refers to a subject exhibiting a statisticallysignificantly lower response to the administration of a statin ascompared to a reference level. The reference level can be, e.g., theaverage response for a population of subjects or the level of theindividual subject at an earlier date. A response to statin treatment isreadily measured by one of skill in the art, e.g., measurement ofcholesterol levels, changes in cholesterol levels, and/or HMG-CoAreductase activity.

As used herein, the term “detectable label” refers to a molecule ormoiety that can be detected, e.g. measured and/or determined to bepresent or absent. Detectable labels can comprise, for example, alight-absorbing dye, a fluorescent dye, or a radioactive label.Detectable labels, methods of detecting them, and methods ofincorporating them into reagents (e.g. antibodies and nucleic acidprobes) are well known in the art.

In some embodiments, detectable labels can include labels that can bedetected by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluorescence, or chemiluminescence, or any other appropriate means.The detectable labels used in the methods described herein can beprimary labels (where the label comprises a moiety that is directlydetectable or that produces a directly detectable moiety) or secondarylabels (where the detectable label binds to another moiety to produce adetectable signal, e.g., as is common in immunological labeling usingsecondary and tertiary antibodies). The detectable label can be linkedby covalent or non-covalent means to the reagent. Alternatively, adetectable label can be linked such as by directly labeling a moleculethat achieves binding to the reagent via a ligand-receptor binding pairarrangement or other such specific recognition molecules. Detectablelabels can include, but are not limited to radioisotopes, bioluminescentcompounds, chromophores, antibodies, chemiluminescent compounds,fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detectable label can be a fluorescentcompound. When the fluorescently label is exposed to light of the properwavelength, its presence can then be detected due to fluorescence. Insome embodiments, a detectable label can be a fluorescent dye molecule,or fluorophore including, but not limited to fluorescein, phycoerythrin,phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™,allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandemconjugates such as phycoerythrin-Cy5™, green fluorescent protein,rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™,rhodamine and derivatives (e.g., Texas red and tetrarhodimineisothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™,6-carboxyfhiorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. In some embodiments, a detectable label can be aradiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and³³P. In some embodiments, a detectable label can be an enzyme including,but not limited to horseradish peroxidase and alkaline phosphatase. Anenzymatic label can produce, for example, a chemiluminescent signal, acolor signal, or a fluorescent signal. Enzymes contemplated for use as adetectable label can include, but are not limited to, malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,catalase, glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. In some embodiments, a detectable label is achemiluminescent label, including, but not limited to lucigenin,luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole,acridinium salt and oxalate ester. In some embodiments, a detectablelabel can be a spectral colorimetric label including, but not limited tocolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads.

In some embodiments, reagents can also be labeled with a detectable tag,such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detectionsystems can also be used, for example, a biotin-streptavidin system. Inthis system, the antibodies immunoreactive (i. e. specific for) with thebiomarker of interest is biotinylated. Quantity of biotinylated antibodybound to the biomarker is determined using a streptavidin-peroxidaseconjugate and a chromagenic substrate. Such streptavidin peroxidasedetection kits are commercially available, e. g. from DAKO; Carpinteria,Calif. A reagent can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the reagent using such metal chelating groupsas diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

As used herein, “authorization number” or “marketing authorizationnumber” refers to a number issued by a regulatory agency upon thatagency determining that a particular medical product and/or compositionmay be marketed and/or offered for sale in the area under the agency'sjurisdiction. As used herein “regulatory agency” refers to one of theagencies responsible for evaluating, e.g, the safety and efficacy of amedical product and/or composition and controlling the sales/marketingof such products and/or compositions in a given area. The Food and DrugAdministration (FDA) in the US and the European Medicines Agency (EPA)in Europe are but two examples of such regulatory agencies. Othernon-limiting examples can include SDA, MPA, MHPRA, IMA, ANMAT, Hong KongDepartment of Health-Drug Office, CDSCO, Medsafe, and KFDA.

As used herein, “injection device” refers to a device that is designedfor carrying out injections, an injection including the steps oftemporarily fluidically coupling the injection device to a person'stissue, typically the subcutaneous tissue. An injection further includesadministering an amount of liquid drug into the tissue and decoupling orremoving the injection device from the tissue. In some embodiments, aninjection device can be an intravenous device or IV device, which is atype of injection device used when the target tissue is the blood withinthe circulatory system, e.g., the blood in a vein. A common, butnon-limiting example of an injection device is a needle and syringe.

As used herein, a “buffer” refers to a chemical agent that is able toabsorb a certain quantity of acid or base without undergoing a strongvariation in pH.

As used herein, “packaging” refers to how the components are organizedand/or restrained into a unit fit for distribution and/or use. Packagingcan include, e.g., boxes, bags, syringes, ampoules, vials, tubes,clamshell packaging, barriers and/or containers to maintain sterility,labeling, etc.

As used herein, “instructions” refers to a display of written, printedor graphic matter on the immediate container of an article, for examplethe written material displayed on a vial containing a pharmaceuticallyactive agent, or details on the composition and use of a product ofinterest included in a kit containing a composition of interest.Instructions set forth the method of the treatment as contemplated to beadministered or performed.

As used herein, a “solid surface” refers to an object suitable for theattachment of biomolecules. Non-limiting examples of a solid surface caninclude a particle (including, but not limited to an agarose or latexbead or particle or a magnetic particle), a bead, a nanoparticle, apolymer, a substrate, a slide, a coverslip, a plate, a dish, a well, amembrane, and/or a grating. The solid surface can include many differentmaterials including, but not limited to, polymers, plastics, resins,polysaccharides, silicon or silica based materials, carbon, metals,inorganic glasses, and membranes.

As used herein, “classification” of a subject, e.g., classification ofthe subject's ancestry refers to determining if the subject hasbiological ancestors who originated in a particular geographical area,and are therefore likely to have particular genetic variants found inthe populations which have historically occupied that area.Classification can comprise, e.g. obtaining information on the subject'sfamily, interviewing the subject or a family member regarding theirbiological family's ancestry, and/or genetic testing. Classification canbe on the basis used for the 1000 Genomes Project, as will be familiarto the skilled person in the art. In some embodiments, the subject canbe classified as being of a particular ancestry if at least thesubject's genome comprises a substantial number of different alleles incommon with other humans of that ancestry (eg, determined by referenceto the 1000 Genomes Project database), for example, at least 10, 20, 30,40, 50 or 100 or more alleles in common. Abbreviations for particularancestral groups are provided in Table 4.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

It will be understood that particular configurations, aspects, examples,clauses and embodiments described herein are shown by way ofillustration and not as limitations of the invention. The principalfeatures of this invention can be employed in various embodimentswithout departing from the scope of the invention. Those skilled in theart will recognize, or be able to ascertain using no more than routinestudy, numerous equivalents to the specific procedures described herein.Such equivalents are considered to be within the scope of this inventionand are covered by the claims. All publications and patent applicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this invention pertains. Allpublications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” The use ofthe term “or” in the claims is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variationof error for the device, the method being employed to determine thevalue, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the following nonlimiting Examples.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising    -   a. selecting a human that is positive for the TOI polymorphic        variant, wherein the TOI in said human is encoded by a        nucleotide sequence comprising an allele having a cumulative        human allele frequency of less than 50% and/or wherein the TOI        in said human is encoded by a nucleotide sequence comprising an        allele having total human genotype frequency of less than 50%;        and    -   b. administering to the human an anti-TOI ligand to target the        TOI in the human to treat or prevent said disease or condition.    -   In an alternative where the TOI is human PCSK9, paragraph 1        provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation as defined herein (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody comprises a VH        domain derived from the recombination of a human VH segment, a        human D gene segment and a human JH segment, the human VH        segment encoding a valine at the amino acid corresponding to        position 5 of SEQ ID NO: 40 and wherein said human comprises (i)        a VH gene segment encoding the framework 1 of SEQ ID NO: 40        and (ii) a nucleotide sequence encoding a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation in SEQ ID NO: 1.    -   In an alternative where the TOI is human PCSK9, paragraph 1        provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation as defined herein (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody comprises a VL        domain derived from the recombination of a human VL segment and        a human JL segment, the human VL segment is a Vλ or Vκ disclosed        herein and wherein said human comprises (i) said VL gene segment        and (ii) a nucleotide sequence encoding a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation in SEQ ID NO: 1.    -   In an alternative where the TOI is human PCSK9, paragraph 1        provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation as defined herein (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody comprises a C        domain encoded by a human CH, Cλ or Cκ gene segment disclosed        herein and wherein said human comprises (i) said C gene segment        and (ii) a nucleotide sequence encoding a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation in SEQ ID NO: 1.-   2. The method of paragraph 1, wherein before step (a) the ligand has    been or is determined as being capable of specifically binding to    said TOI variant.-   3. The method of paragraph 1 or 2, comprising determining that the    human is positive for the TOI polymorphic variant, optionally    wherein the step of determining comprises determining that the human    is positive for a nucleotide variant encoding said TOI variant.-   4. The method of paragraph 3, wherein the step of determining    comprises assaying a biological sample obtained from said human for    a nucleotide polymorphism encoding said TOI polymorphic variant.-   5. The method of paragraph 3 or 4, wherein the step of determining    comprises assaying a biological sample obtained from said human for    a protein corresponding to the TOI polymorphic variant.-   6. The method of any preceding paragraph, wherein said frequency is    less than 15%.-   7. The method of any preceding paragraph, wherein said frequency is    less than 10%.-   8. The method of any preceding paragraph, wherein the ligand is    capable of specifically binding to two or more different TOI    variants, each being encoded by a nucleotide sequence comprising an    allele having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50%.-   9. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising    -   a. selecting a human that is negative for a variant nucleotide        sequence comprising an allele having a cumulative human allele        frequency of less than 50% and/or having a total human genotype        frequency of less than 50%; or that the human is negative for a        TOI variant encoded by a nucleotide sequence comprising the        allele having a cumulative human allele frequency of less than        50% and/or having a total human genotype frequency of less than        50%; and    -   b. administering to the human an anti-TOI ligand to target the        TOI variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is a variant encoded by        a nucleotide sequence comprising an allele having a cumulative        human allele frequency of more than 50% and/or having a total        human genotype frequency of more than 50%.-   10. The method of paragraph 9, comprising determining that the human    is positive for the TOI polymorphic variant, optionally wherein the    determining comprises that the human has been or is phenotyped as    positive for the most frequent TOI variant or genotyped for the    nucleotide sequence thereof-   11. The method of paragraph 10, wherein determining comprises    assaying for the nucleotide sequence to determine the presence of    said allele.-   12. The method of paragraph 11, wherein the assaying comprises    nucleic acid amplification.-   13. The method of paragraph 11 or 12, wherein the assaying comprises    hybridization, sequencing, or next generation sequencing.-   14. The method of any of paragraphs 11-13, further comprising the    step of obtaining a biological sample from the human.-   15. The method of any one of paragraphs 9-14, wherein the ligand has    been or is determined as being capable of specifically binding to    the most frequent TOI variant.-   16. The method of any one of paragraphs 9-15, wherein the ligand has    been or is determined as being substantially incapable of    neutralising or inhibiting said TOI variant.-   17. The method of any one of paragraphs 9-16, wherein the ligand is    capable of specifically binding to the most frequent TOI variant.-   18. The method of any one of paragraphs 9-17, wherein the ligand is    capable of specifically binding to two or more different TOI    variants, each being encoded by a nucleotide sequence comprising an    allele having a cumulative human allele frequency of more than 50%.-   19. The method of any preceding paragraph, wherein said TOI    polymorphic variant has been or is determined as being present in at    least two different human ethnic populations.-   20. The method of any preceding paragraph, wherein said cumulative    human allele frequency is the frequency in a database of    naturally-occurring sequences sampled from at least 15 different    human ethnic populations and comprising at least 1000 sequences.-   21. The method of any of the preceding paragraphs, wherein the    ligand is an antibody, antibody fragment or an affibody.-   22. The method of any of the preceding paragraphs, wherein the    ligand comprises a nucleotide sequence that specifically hybridises    to a TOI nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or having a    total human genotype frequency of less than 50% or an RNA transcript    thereof; and/or the ligand comprises a nucleotide sequence that    comprises at least 10 contiguous nucleotides of a nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50% or    is an antisense sequence thereof-   23. The method of any of the preceding paragraphs, wherein the    genome of said human comprises an allele having a cumulative human    allele frequency of less than 50% and the allele is found in at    least 2 different ethnic populations.-   24. A composition comprising a ligand capable of binding a target of    interest encoded by a nucleotide sequence comprising an allele    having a cumulative human allele frequency of less than 50% and the    allele is found in at least 2 different ethnic populations and    optionally a pharmaceutically acceptable carrier and optionally a    label or instructions for use to treat and/or prevent said disease    or condition in a human; optionally wherein the label or    instructions comprise a marketing authorisation number issued by a    regulatory authority.-   25. A kit for treating or preventing a condition or disease mediated    by a target of interest as recited in any preceding paragraph, the    kit comprising a ligand capable of specifically binding a target of    interest encoded by a nucleotide sequence comprising an allele    having a cumulative human allele frequency of less than 50% and the    allele is found in at least 2 different ethnic populations; and    optionally in combination with a label or instructions for use to    treat and/or prevent said disease or condition in a human;    optionally wherein the label or instructions comprise a marketing    authorisation number issued by a regulatory agency; optionally    wherein the kit comprises an injection pen or IV container that    comprises the ligand.-   26. The composition of paragraph 24 or the kit of paragraph 25,    wherein the regulatory agency is FDA or EMA.-   27. A method of producing an anti-human TOI antibody binding site,    the method comprising obtaining a plurality of anti-TOI antibody    binding sites, screening the antibody binding sites for binding to a    TOI comprising an amino acid sequence encoded by a nucleotide    sequence comprising an allele having a cumulative human allele    frequency of less than 50% and/or a total human genotype frequency    of less than 50%, or to a peptide thereof that comprises an amino    acid variation from the corresponding sequence encoded by the    TOI-encoding nucleotide sequence comprising an allele having the    highest cumulative human allele frequency and/or the highest total    human genotype frequency, and isolating an antibody binding site    that binds in the screening step.-   28. A method of producing an anti-human TOI antibody, the method    comprising immunising a non-human vertebrate with a TOI comprising    an amino acid sequence encoded by a nucleotide sequence comprising    an allele having a cumulative human allele frequency of less than    50% and/or a total human genotype frequency of less than 50%, or to    a peptide thereof that comprises an amino acid variation from the    corresponding sequence encoded by the TOI-encoding nucleotide    sequence comprising an allele having the highest cumulative human    allele frequency and/or the highest total human genotype frequency,    and isolating an antibody that binds a TOI comprising an amino acid    sequence encoded by a TOI nucleotide sequence comprising an allele    having a cumulative human allele frequency of less than 50% and/or a    total human genotype frequency of less than 50%, and optionally    producing a TOI-binding fragment or derivative of the isolated    antibody.-   29. The method of paragraph 28, wherein the non-human vertebrate is    a mouse or a rat.-   30. The method of paragraph 29 or 30, comprising the step of    obtaining a nucleic acid encoding the antibody, fragment, derivative    or binding site and optionally inserting the nucleic acid in an    expression vector.-   31. A kit for TOI genotyping a human, wherein the kit comprises a    nucleic acid comprising a nucleotide sequence that specifically    hybridises to a TOI nucleotide sequence selected having a cumulative    human allele frequency of less than 50% and/or a total human    genotype frequency of less than 50% or an RNA transcript thereof;    and/or the nucleic acid comprises a nucleotide sequence that    comprises at least 10 contiguous nucleotides of a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50% or is an    antisense sequence thereof-   32. A kit for TOI genotyping or phenotyping a human, wherein the kit    comprises a ligand capable of binding a target of interest encoded    by a nucleotide sequence comprising an allele having a cumulative    human allele frequency of less than 50% or an antibody, fragment or    derivative produced by the method of any one of paragraphs 28 to 30.-   33. The kit of paragraph 32, wherein the allele is found in at least    2 different ethnic populations.-   34. Use of an anti-TOI ligand that specifically binds a human TOI    comprising an amino acid sequence encoded by a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, in the    manufacture of a medicament for treating and/or preventing a    TOI-mediated disease or condition in a human whose genome comprises    a TOI nucleotide sequence having a cumulative human allele frequency    of less than 50% and/or having a total human genotype frequency of    less than 50%.-   35. Use of an anti-TOI ligand that specifically binds a human TOI    comprising an amino acid sequence encoded by a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, in the    manufacture of a medicament for targeting said TOI in a human to    treat and/or prevent a disease or condition mediated by TOI.-   36. A method of targeting a Target of Interest (TOI) for treating    and/or preventing a TOI-mediated disease or condition in a human,    the method comprising administering an anti-TOI ligand to a human    comprising a TOI nucleotide sequence comprising an allele selected    as having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, whereby a    TOI encoded by said nucleotide sequence is targeted.-   37. The method of paragraph 36, wherein the method comprises    targeting a human TOI comprising an amino acid sequence with said    ligand to treat and/or prevent said disease or condition in said    human, wherein said amino acid sequence is encoded by a nucleotide    sequence comprising an allele having a cumulative human allele    frequency of less than 50% and/or a total human genotype frequency    of less than 50%.-   38. A method of Target of Interest (TOI) genotyping a nucleic acid    sample of a human, the method comprising assaying in the sample the    presence of a TOI nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or having a    total human genotype frequency of less than 50%.-   39. A method of Target of Interest (TOI) typing a protein sample of    a human, the method comprising assaying the sample the presence of a    TOI amino acid sequence encoded by a TOI nucleotide sequence    comprising an allele having a cumulative human allele frequency of    less than 50% and/or having a total human genotype frequency of less    than 50%.-   40. The method of paragraph 38 or 39, comprising obtaining a sample    of serum, blood, faeces, hair, urine or saliva from a human, whereby    the nucleic acid or protein sample is obtained for use in the step    of assaying said sequence.-   41. The method of any one of paragraphs 38-40, comprising using a    ligand capable of targeting a nucleic acid sequence comprising an    allele having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50% or a    ligand capable of specifically binding the TOI encoded by said    nucleic acid sequence to carry out said identifying step.-   42. A diagnostic kit comprising a ligand that is capable of binding    a human Target of Interest (TOI) comprising an amino acid sequence    encoded by a TOI nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or a total    human genotype frequency of less than 50% and instructions for    carrying out the method of any one of paragraphs 38-41.-   43. The diagnostic kit wherein the ligand is selected from an    antibody, antibody fragment, antibody portion, affybody,    oligonucleotide, modified oligonucleotide, antisense    oligonucleotide, siRNA, and microRNA.-   44. A diagnostic kit comprising a nucleic acid probe comprising a    nucleotide sequence that specifically hybridises a Target of    Interest (TOI) nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or a total    human genotype frequency of less than 50% or an RNA transcript    thereof and instructions for carrying out the method of paragraph 38    or 39.-   45. The method, ligand, composition, kit or use of any preceding    paragraph, wherein the TOI is encoded by a nucleotide sequence    having a cumulative human allele frequency from 1 to 10% and/or a    total human genotype frequency from 1 to about 15% or from 1 to 15%.-   46. The method, ligand, composition, kit or use of any preceding    paragraph wherein the TOI is a human TOI selected from Table 5;    optionally for treating and/or preventing a corresponding disease or    condition as set out in Table 5.-   47. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising administering to the    human determined to be positive for the TOI polymorphic variant,    wherein the TOI in said human is encoded by a nucleotide sequence    comprising an allele having a cumulative human allele frequency of    less than 50% and/or wherein the TOI in said human is encoded by a    nucleotide sequence comprising an allele having total human genotype    frequency of less than 50% an anti-TOI ligand to target the TOI in    the human to treat or prevent said disease or condition.-   48. The method of paragraph 47, wherein the anti-TOI ligand is    selected from an antibody, an antibody portion, an antibody    fragment, an affibody, an antisense oligonucleotide, an siRNA, and a    microRNA.

Additional Tailoring of Ligands to Genotype and/or Phenotype of theHuman Patient

As described herein, the present invention contemplates ligands (eg,antibodies and fragments) whose binding site specificities have beenmatched to one or more variant human TOIs (eg, PCSK9 or IL6R).Additionally or alternatively (and as further illustrated in thenon-limiting Examples below), an optional aspect of the inventionprovides for matching of other features of the ligand to the patient'sgenotype or phenotype. In this respect, for example, the inventionincludes the ability to match amino acid sequence variation in a humanpatient to one or more ligand sequences or domains outside of thebinding sites. For example, where the ligand comprises or consists of ahuman TOI-binding antibody or an anti-human TOI receptor Fc fusion, thisaspect of the invention provides for more tailored matching of one ormore constant region domains (eg, the Fc) to the patient genotype orphenotype. Additionally or alternatively, it is contemplated thatsequence variation in the binding site can be similarly matched to thepatient's genotype or phenotype. The present inventor has done this byconsidering the SNP occurrences in sequences encoding one or more partsof the ligand, eg, SNP occurrences in one, more or all of the genesegments from which the variable domain(s) and/or constant regiondomain(s) are derived. The inventor realised that it would be desirableto match the ligand to one or more corresponding variant SNPs found inthe patient to be treated therapeutically and/or prophylactically.Matching could involve designing the ligand specifically for a patientof known phenotype and/or genotype, or matching could involve choosing aligand by determining that there is correspondence between variation inthe patient's phenotype or genotype with the variation in the ligandamino acid and/or corresponding nucleotides.

A key consideration for the inventor was the desire to promotecompatibility of the ligand with the patient's body, and in particular,the possible patient immune system responses to administered ligands.For example, it has been observed that human patients receiving human orhumanised antibody drugs may mount an immune response against theincoming antibody (a so-called HAHA response) which results in thepatient producing anti-drug antibodies as a result of the patient'simmune system recognising the drug as foreign. For example, studies havesuggested that some patients receiving HUMIRA™ (adalimumab), currentlythe biggest selling antibody medicine, mount a HAHA immune responseagainst the medicine, and this may impact treatment adversely. Referenceis made to JAMA. 2011; 305(14):1460-1468. doi:10.1001/jama.2011.406:“Development of Antidrug Antibodies Against Adalimumab and AssociationWith Disease Activity and Treatment Failure During Long-term Follow-up”,GM Bartelds et al. The authors concluded that results of this studyshowed that development of antidrug antibodies was associated with anegative outcome of adalimumab treatment in human RA patients. It wasreported that not only did patients with anti-adalimumab antibodiesdiscontinue treatment more often and earlier than patients withoutanti-adalimumab antibodies, they also had a higher disease activityduring treatment and only rarely came into remission. In addition,reportedly the data showed that two-thirds of the anti-adalimumabantibody—positive patients developed these antibodies in the first 28weeks of treatment and that the presence of anti-adalimumab antibodiessubstantially influenced serum adalimumab concentrations.

This HAHA theme is, therefore, a significant concern and this has beenconsidered by the regulatory authorities. For example, the EuropeanMedicines Agency (EMA) has issued a “Guideline on immunogenicityassessment of monoclonal antibodies intended for in vivo clinical use”(available on the world wide web atema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/06/WC500128688.pdf;EMA/CHMP/BMWP/86289/2010, addendum to EMEA/CHMP/BMWP/14327/2006), whichcame into force on 1^(st) Dec. 2012. As such, it is good practice forresearchers to identify and assess risk of anti-antibody drug occurrenceand effects.

The present aspect of the invention, by more closely tailoring theligand itself (as well as its specificity) to the patient, helps toaddress these considerations when designing and administering anti-humanTOI medicines for treating and/or preventing human TOI-related diseasesand conditions.

The inventor also considered the desirability to tailor the variation inthe ligand constant region (eg, for an antibody or Fc-containingligand), mindful that then the constant region being administered to thepatient would be tuned to the various components, such as patient's Fcreceptors, that would interact with the constant region in the patient.Good Fc/Fc receptor interactions can be important for drug recycling(via the FcRn) to provide for useful half-lives in vivo or for use incell killing, eg, for cancer indications. In this way it is possible,therefore, to tune the effector function of the constant region (eg, Fc)to the patient more closely, to promote efficacy. For example, moreefficacious drugs are desirable for better patient treatment and mayprovide the possibility of lowered dosing and/or dosing frequencies.

Thus, in examples of this aspect, the invention provides the following(set out as clauses):—

-   1. The ligand, method, use, kit or composition of the invention,    wherein    -   (i) the ligand (eg, antibody or fragment) comprises        -   (c) a variable domain that is encoded by a human V region            nucleotide sequence, wherein the V nucleotide sequence is            derived from recombination of human VH, D and JH gene            segments or human VL and JL gene segments; or        -   (d) a constant region domain encoded by a C region gene            segment;        -   Wherein a first gene segment of said gene segments of (a),            or said C region gene segment of (b) comprises a first            single nucleotide polymorphism (SNP) encoding a first amino            acid polymorphism; and    -   (ii) the genome of said human comprises said first SNP or        wherein said human expresses (a′) an antibody variable domain        comprising said first amino acid polymorphism or (b′) an        antibody constant domain comprising said first amino acid        polymorphism.-   2. The ligand, method, use, kit or composition of clause 1, wherein    blood of said human comprises substantially no antibodies that    specifically bind to the domain comprising said first amino acid    polymorphism as determined in an in vitro binding assay.-   3. The ligand, method, use, kit or composition of clause 2, wherein    SPR is used to carry out said assay.    -   In an alternative, ELISA is used.-   4. The ligand, method, use, kit or composition of any one of clauses    1 to 3, wherein the genome of said human comprises said first gene    segment (when (a) applies) or said C region gene segment (when (b)    applies).-   5. The ligand, method, use, kit or composition of any one of clauses    1 to 4, wherein said first segment or a second segment of said    segments of (a), or said C region gene segment of (b), comprises a    second SNP encoding a second amino acid polymorphism; and wherein    the genome of said human comprises said second SNP or wherein said    human expresses (a″) an antibody variable domain comprising said    second amino acid polymorphism or (b″) an antibody constant region    domain comprising said first and second amino acid polymorphisms.-   6. The ligand, method, use, kit or composition of clause 5, wherein    said human expresses an antibody variable domain comprising said    first and second amino acid polymorphisms.-   7. The ligand, method, use, kit or composition of clause 5 or 6,    wherein the first and second SNPs of said genome are comprised by    the same antibody gene segment.    -   For example, the first and second SNPs of the genome are        comprised by an IGHG1*01 gene segment and said first segment        of (a) is an IGHG1*01 gene segment.    -   For example, the first and second SNPs of the genome are        comprised by an IGHG2*01 gene segment and said first segment        of (a) is an IGHG2*01 gene segment.-   8. The ligand, method, use, kit or composition of any one of clauses    1 to 7, wherein each SNP is a variable region gene segment SNP.-   9. The ligand, method, use, kit or composition of any one of clauses    1 to 7, wherein each SNP is a constant region gene segment SNP, eg    each SNP is a gamma-1 constant region gene segment SNP, or a gamma-2    constant region gene segment SNP, or a gamma-3 constant region gene    segment SNP or a gamma-4 constant region gene segment SNP.-   10. The ligand, method, use, kit or composition of clause 9, wherein    the first SNP is a CH1, CH2, CH3 or CH4 gene segment SNP and/or the    second SNP is a CH1, CH2, CH3 or CH4 gene segment SNP.-   11. The ligand, method, use, kit or composition of any one of    clauses 1 to 8, wherein each SNP is a variable domain SNP, eg, a VH    domain SNP, or a Vκ domain SNP, or a Vλ SNP.-   12. The ligand, method, use, kit or composition of any one of    clauses 1 to 11, wherein said constant region domain of (b) is    comprised by an antibody Fc region.-   13. The ligand, method, use, kit or composition of any one of    clauses 1 to 12, wherein the ligand (eg, antibody or fragment) has    been determined to specifically bind one or more human TOI variants    as disclosed herein, for example, with a KD of 1 nM or less (eg, 100    or 10 pM or less) as determined by SPR.-   14. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 13), wherein the ligand    comprises or consists of an antibody or fragment that comprises a    human antibody variable domain derived from the recombination of a    human V gene segment and a human J gene segment (and optionally a    human D gene segment when the variable domains are VH domains); and    wherein the genome of the human comprises said human V gene segment    and/or the human expresses antibodies comprising antibody variable    domains derived from the recombination of said human V gene segment    and a human J gene segment (and optionally a human D gene segment).    -   In an example, the V gene segment is any of the V gene segments        disclosed in WO2013041844, a 1000 Genomes database and/or        www.imgt.org, the disclosures of which (including disclosure        relating to sequence) is explicitly incorporated herein by        reference for use in the present invention.-   15. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 14), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human heavy chain constant    domain encoded by a first constant region nucleotide sequence; and    wherein the genome of the human comprises a heavy chain constant    region nucleotide sequence that is identical to said first constant    region nucleotide sequence and/or the human expresses antibodies    comprising said human constant domain.-   16. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 15), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH1    domain encoded by a CH1 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH1 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH1 domain.-   17. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 16), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH2    domain encoded by a CH2 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH2 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH2 domain.-   18. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 17), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH3    domain encoded by a CH3 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH3 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH3 domain.-   19. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 18), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH4    domain encoded by a CH4 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH4 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH4 domain.-   20. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 19), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain Fc    region encoded by a Fc nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said Fc nucleotide sequence    and/or the human expresses antibodies comprising said human gamma Fc    region.-   21. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein said human gamma heavy chain is a human    gamma-1 heavy chain-   22. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein said human gamma heavy chain is a human    gamma-2 heavy chain-   23. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein ligand comprises a human IGHG1*01 gamma-1    heavy chain constant region.-   24. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein ligand comprises a human IGHG2*01 gamma-1    heavy chain constant region.-   25. The ligand, method, use, kit or composition of any one of    clauses 15 to 24, wherein the human has been or is genotyped as    positive for said heavy chain constant region nucleotide sequence.-   26. The ligand, method, use, kit or composition of clause 23,    wherein the human has been or is genotyped as positive for human    IGHG1*01 nucleotide sequence.-   27. The ligand, method, use, kit or composition of clause 24,    wherein the human has been or is genotyped as positive for human    IGHG2*01 nucleotide sequence.-   28. The ligand, method, use, kit or composition of any one of    clauses 16 to 24, wherein the human has been or is phenotyped as    positive for said gamma heavy chain constant domain, CH1, CH2, CH3,    CH4 or Fc.-   29. The ligand, method, use, kit or composition of clause 28, (i)    when dependent from clause 23, wherein the human has been or is    phenotyped as positive for a human IGHG1*01 gamma heavy chain    constant domain, CH1, CH2, CH3, CH4 or Fc or (ii) when dependent    from clause 24, wherein the human has been phenotyped as positive    for a human IGHG2*01 gamma heavy chain constant domain, CH1, CH2,    CH3, CH4 or Fc.-   30. The method or use of any one of clauses 16 to 24 and 26 to 29,    comprising genotyping the human as positive for said gamma heavy    chain constant region nucleotide sequence, eg, positive for said    gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc    nucleotide sequence; positive for said human IGHG1*01 gamma heavy    chain constant region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence;    or positive for said human IGHG2*01 gamma heavy chain constant    region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence.-   31. The method or use of any one of clauses 16 to 24 and 26 to 30,    comprising phenotyping the human as positive for said gamma heavy    chain constant region, eg, positive for said gamma heavy chain    constant domain, CH1, CH2, CH3, CH4 or Fc; positive for said human    IGHG1*01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or    Fc; or positive for said human IGHG2*01 gamma heavy chain constant    domain, CH1, CH2, CH3, CH4 or Fc.

Examples of Tailored Ligands

The inventor analysed amino acid variability and distribution amongstlarge representative human samples. The result of the analysis forexample antibody gene segments is shown in Table 9.

In a first example, the inventor identified the possibility ofaddressing the rarer IGH-gamma-1 SNPs 204D (observed cumulativefrequency of 0.296) and 206L (observed cumulative frequency of 0.283)individually or in combination. These residues are part of the CH3domain, and as such they form part of antibody Fc regions. Thus,matching of these CH3 variations with the patient is especiallybeneficial for reasons as discussed above. Thus, this example providesaspects set out in the following clauses.

-   32. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 31), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human gamma-1 heavy chain    constant region that comprises an Asp corresponding to position 204    of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID    NO: 42 and wherein the genome of the human comprises a gamma-1 heavy    chain constant region nucleotide sequence that encodes such an Asp    or Leu or the human expresses antibodies comprising human gamma-1    constant regions comprising such an Asp or Leu.    -   The skilled person will be familiar with techniques for        determining genome sequences of a human, eg, by using a sample        containing genomic DNA and/or RNA, sequencing and comparing        using bioinformatics or other computer tools to compare the        sampled sequence with sequences of human alleles (eg, as shown        in the IMGT, 100 genomes or other database as disclosed herein).        In an example, the sample is a blood or saliva or cheek swab        sample.-   33. The ligand, method, use, kit or composition of clause 32,    wherein the ligand comprises a human gamma-1 heavy chain constant    region that comprises an Asp corresponding to position 204 of SEQ ID    NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42.-   34. The ligand, method, use, kit or composition of clause 32 or 33,    wherein the genome of the human comprises a gamma-1 heavy chain    constant region nucleotide sequence that encodes such an Asp and Leu    or the human expresses antibodies comprising human gamma-1 constant    regions comprising such an Asp and Leu.-   35. The ligand, method, use, kit or composition of clause 32, 33 or    34, wherein the ligand comprises a human IGHG1*01 gamma-1 heavy    chain constant region, eg, an Fc, CH1, CH2 and/or CH3 domain encoded    by human IGHG1*01.-   36. The ligand, method, use, kit or composition of any one of    clauses 32 to 35, wherein the genome of the human comprises a human    IGHG1*01 nucleotide sequence or the human expresses antibodies    comprising human constant domains encoded by a human IGHG1*01    nucleotide sequence.-   37. The ligand, method, use, kit or composition of any one of    clauses 32 to 36, wherein the ligand comprises a hinge region    encoded by human IGHG1*01.-   38. The ligand, method, use, kit or composition of any one of    clauses 32 to 37, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises heavy chains that comprise    SEQ ID NO: 61.-   39. The ligand, method, use, kit or composition of any one of    clauses 32 to 38, wherein the human is of European ancestry.    -   As shown in Table 9, 204D and 206L are found in such humans.-   40. The ligand, method, use, kit or composition of any one of    clauses 32 to 39, wherein the human has been or is genotyped as    positive for said Asp and/or Leu.-   41. The ligand, method, use, kit or composition of any one of    clauses 32 to 40, wherein the human has been or is genotyped as    positive for human IGHG1*01.-   42. The ligand, method, use, kit or composition of any one of    clauses 32 to 41, wherein the human has been or is phenotyped as    positive for a human IGHG1*01 CH3.-   43. The method or use of any one of clauses 32 to 42, comprising    selecting a said human whose genome comprises a codon(s) encoding    said Asp and/or Leu; comprises human IGHG1*01; or comprises a human    IGHG1*01 CH3.-   44. The method or use of any one of clauses 32 to 43, comprising    selecting a said human whose phenotype comprises said Asp and/or    Leu; a human IGHG1*01 region; or a human IGHG1*01 CH3.    -   44a. The ligand, method, use, kit or composition of any one of        clauses 32 to 44, wherein the human expresses antibodies        comprising human gamma-1 constant regions comprising such an Asp        and Leu.

In a second example, the inventor identified the possibility ofaddressing IGH-gamma-2 SNPs. This included consideration of Fc regionvariation—in this respect, the inventor focused on positions 161 and 257which are in the Fc region. Thus, this example provides aspects set outin the following clauses.

-   45. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 31), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human gamma-2 heavy chain    constant region that comprises an amino acid selected from the group    consisting of a Pro corresponding to position 72 of SEQ ID NO: 44,    an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe    corresponding to position 76 of SEQ ID NO: 44, a Val corresponding    to position 161 of SEQ ID NO: 44 and an Ala corresponding to    position 257 of SEQ ID NO: 44; and wherein the genome of the human    comprises a gamma-2 heavy chain constant region nucleotide sequence    that encodes such a selected amino acid or the human expresses    antibodies comprising human gamma-2 constant regions comprising such    a selected amino acid.-   46. The ligand, method, use, kit or composition of clause 45,    wherein the ligand comprises a human gamma-2 heavy chain constant    region that comprises (i) a Pro corresponding to position 72 of SEQ    ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a    Phe corresponding to position 76 of SEQ ID NO: 44 and    optionally (ii) a Val corresponding to position 161 of SEQ ID NO: 44    and/or an Ala corresponding to position 257 of SEQ ID NO: 44; and    wherein the genome of the human comprises a gamma-2 heavy chain    constant region nucleotide sequence that encodes such amino acids    of (i) or the human expresses antibodies comprising human gamma-2    constant regions comprising such amino acids of (i).    -   This example focuses on CH1 variation.-   47. The ligand, method, use, kit or composition of clause 45 or 46,    wherein the ligand comprises a human gamma-2 heavy chain constant    region that comprises (i) a Val corresponding to position 161 of SEQ    ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44    and optionally (ii) an amino acid selected from the group consisting    of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn    corresponding to position 75 of SEQ ID NO: 44 and a Phe    corresponding to position 76 of SEQ ID NO: 44; and wherein the    genome of the human comprises a gamma-2 heavy chain constant region    nucleotide sequence that encodes such amino acids of (i) or the    human expresses antibodies comprising human gamma-2 constant regions    comprising such amino acids of (i).    -   This example focuses on Fc variation.-   48. The ligand, method, use, kit or composition of any one of    clauses 45 to 47, wherein the ligand comprises a human IGHG2*01    gamma-2 heavy chain constant region, eg, an Fc, CH1, CH2 and/or CH3    domain encoded by human IGHG2*01.-   49. The ligand, method, use, kit or composition of any one of    clauses 45 to 48, wherein the genome of the human comprises a human    IGHG2*01 nucleotide sequence or the human expresses antibodies    comprising human constant domains encoded by a human IGHG2*01    nucleotide sequence.-   50. The ligand, method, use, kit or composition of any one of    clauses 45 to 49, wherein the ligand comprises a hinge region    encoded by human IGHG2*01.-   51. The ligand, method, use, kit or composition of any one of    clauses 45 to 50, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises heavy chains that comprise    SEQ ID NO: 63 or 65.-   52. The ligand, method, use, kit or composition of any one of    clauses 45 to 51, wherein the human is of European, African    American, or European American ancestry.-   53. The ligand, method, use, kit or composition of any one of    clauses 45 to 52, wherein the human has been or is genotyped as    positive for one, more or all of said Pro, Asn, Phe, Val and Ala.-   54. The ligand, method, use, kit or composition of any one of    clauses 45 to 53, wherein the human has been or is genotyped as    positive for human IGHG2*01.-   55. The ligand, method, use, kit or composition of any one of    clauses 45 to 54, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH1.-   56. The ligand, method, use, kit or composition of any one of    clauses 45 to 55, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH2.-   57. The ligand, method, use, kit or composition of any one of    clauses 45 to 56, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH3.-   58. The method or use of any one of clauses 45 to 57, comprising    selecting a said human whose genome comprises a codon(s) encoding    one, more or all of said Pro, Asn, Phe, Val and Ala; comprises human    IGHG2*01; or comprises a human IGHG2*01 CH1, CH2 and/or CH3.-   59. The method or use of any one of clauses 45 to 58, comprising    selecting a said human whose phenotype comprises one, more or all of    said Pro, Asn, Phe, Val and Ala; a human IGHG2*01 region; or a human    IGHG2*01 CH1, CH2 and/or CH3.-   60. The ligand, method, use, kit or composition of any one of    clauses 45 to 59, wherein the human expresses antibodies comprising    human gamma-2 constant regions comprising such a Pro, Asn, Phe, Val    and Ala.

In a third example, the inventor addressed human kappa constant regionvariation. Thus, the present aspect of the invention also provides thefollowing.

-   61. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 60), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human kappa light chain constant    region that comprises a Val corresponding to position 84 of SEQ ID    NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50; and    wherein the genome of the human comprises a kappa light chain    constant region nucleotide sequence that encodes such a Val or Cys    or the human expresses antibodies comprising human kappa light chain    constant regions comprising such a Val or Cys.-   62. The ligand, method, use, kit or composition of clause 61,    wherein the ligand comprises a human kappa light chain constant    region that comprises a Val corresponding to position 84 of SEQ ID    NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50.-   63. The ligand, method, use, kit or composition of clause 61 or 62,    wherein the genome of the human comprises a kappa light chain    constant region nucleotide sequence that encodes such a Val and Cys    or the human expresses antibodies comprising human kappa constant    regions comprising such a Val and Cys.-   64. The ligand, method, use, kit or composition of any one of    clauses 61 to 63, wherein the antibody or fragment comprises a human    IGKC*01 kappa light chain constant region.-   65. The ligand, method, use, kit or composition of any one of    clauses 61 to 64, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises light chains that comprise    SEQ ID NO: 62 or 66.-   66. The ligand, method, use, kit or composition of any one of    clauses 61 to 65, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises a light chain variable    domain derived from recombination of a human Vκ gene segment and a    human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ    ID NO: 57).-   67. The ligand, method, use, kit or composition of any one of    clauses 61 to 66, wherein the human has been or is phenotyped as    positive for said Val and/or Cys.-   68. The ligand, method, use, kit or composition of any one of    clauses 61 to 67, wherein the human has been or is genotyped as    positive for human IGKC*01.-   69. The ligand, method, use, kit or composition of any one of    clauses 61 to 68, wherein the human has been or is phenotyped as    positive for a human IGKC*01 domain.-   70. The method or use of any one of clauses 61 to 69, comprising    selecting a said human whose genome comprises a codon(s) encoding    said Val and/or Cys; or comprises human IGKC*01.-   71. The method or use of any one of clauses 61 to 70, comprising    selecting a said human whose phenotype comprises such a Val and/or    Cys; or comprises a human IGKC*01 domain.-   72. The ligand, method, use, kit or composition of any one of    clauses 61 to 71, wherein the human expresses antibodies comprising    human kappa constant domains comprising such a Val and Cys, eg,    expresses human IGKC*01 constant domains.

In a fourth example, the inventor addressed human lambda constant regionvariation. Thus, this example provides aspects set out in the followingclauses.

-   73. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 60), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human IGLC2*01 light chain    constant region; and wherein the genome of the human comprises a    human IGLC2*01 nucleotide sequence or the human expresses antibodies    comprising human light chain IGLC2*01 constant regions.-   74. The ligand, method, use, kit or composition of clause 73,    wherein the antibody comprises light chains that comprise SEQ ID NO:    64.-   75. The ligand, method, use, kit or composition of clause 73 or 74,    wherein the human has been or is genotyped as positive for human    IGLC2*01.-   76. The ligand, method, use, kit or composition of any one of    clauses 73 to 75, wherein the human has been or is phenotyped as    positive for a human IGLC2*01 domain.-   77. The method or use of any one of clauses 73 to 76, comprising    selecting a said human whose genome comprises human IGLC2*01.-   78. The method or use of any one of clauses 73 to 77, comprising    selecting a said human whose phenotype comprises a human IGLC2*01    domain.-   79. The ligand, method, use, kit or composition of any one of    clauses 73 to 78, wherein the human expresses antibodies comprising    human lambda IGLC2*01 constant domains.

In a fifth example, the inventor addressed human heavy chain variableregion variation. Thus, this example provides aspects set out in thefollowing clauses.

-   80. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 79), wherein the ligand    comprises or consists of an antibody or fragment, wherein the    antibody or fragment comprises a VH domain that is derived from the    recombination of a human VH gene segment, a human D gene segment and    a human JH gene segment, wherein the VH gene segment is selected    from the group consisting of (i) IGHV1-18*01 and the genome of the    human comprises a human IGHV1-18*01 nucleotide sequence or the human    expresses antibodies comprising variable domains derived from the    recombination of human IGHV1-18*01; or (ii) IGVH1-46*01 and the    genome of the human comprises a human IGHV1-46*01 nucleotide    sequence or the human expresses antibodies comprising variable    domains derived from the recombination of human IGHV1-46*01.-   81. The ligand, method, use, kit or composition of clause 80,    wherein the antibody or fragment comprises a one more or all of a    CH1 domain, CH2 domain, CH3 domain, hinge or Fc encoded by human    IGHG2*01.-   82. The ligand, method, use, kit or composition of clause 80 or 81,    wherein the antibody or fragment comprises heavy chains that    comprise SEQ ID NO: 63 or 65.-   83. The ligand, method, use, kit or composition of any one of    clauses 80 to 82, wherein the human has been or is genotyped as    positive for said selected VH gene segment, positive for human    IGHV1-18*01 or IGVH1-46*01.-   84. The method or use of any of clauses 80 to 83, comprising    genotyping the human as positive for said selected VH gene segment,    eg, positive for human IGHV1-18*01 or IGVH1-46*01.

In a sixth example, the inventor addressed human light chain variableregion variation. Thus, this example provides aspects set out in thefollowing clauses.

-   85. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 84), wherein the ligand    comprises or consists of an antibody or fragment, wherein the    antibody or fragment comprises a VL domain that is derived from the    recombination of a human VL gene segment and a human JL gene    segment, wherein the VL gene segment is selected from the group    consisting of (i) IGKV4-1*01 and the genome of the human comprises a    human IGKV4-1*01 nucleotide sequence or the human expresses    antibodies comprising variable domains derived from the    recombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome    of the human comprises a human IGLV2-14*01 nucleotide sequence or    the human expresses antibodies comprising variable domains derived    from the recombination of human IGLV2-14*01; or (iii) IGKV1-13*02    and the genome of the human comprises a human IGKV1-13*02 nucleotide    sequence or the human expresses antibodies comprising variable    domains derived from the recombination of human IGKV1-13*02.-   86. The ligand, method, use, kit or composition of clause 85,    wherein the antibody comprises light chains that comprise SEQ ID NO:    62, 64 or 66.-   87. The ligand, method, use, kit or composition of clause 85 or 86,    wherein the antibody or fragment comprises a light chain variable    domain derived from recombination of a human Vκ gene segment and a    human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ    ID NO: 57; wherein (i) or (iii) applies.-   88. The ligand, method, use, kit or composition of any one of    clauses 85 to 87, wherein the human has been or is genotyped as    positive for said selected VL gene segment, eg, positive for human    IGKV4-1*01, IGLV2-14*01 or IGKV1-13*02.-   89. The method or use of clause 88, comprising genotyping the human    as positive for said selected VL gene segment, eg, genotyping the    human as positive for human IGKV4-1*01, IGLV2-14*01 or IGKV1-13*02.-   90. The ligand, method, use, kit or composition of any one of    clauses 1 to 89, wherein the ligand (eg, antibody or fragment) binds    said human TOI with a dissociation constant (Kd) of 1 nM or less as    determined by SPR, (eg, 100, 10 or 1 pM or less).

EXAMPLES Example 1 Rare PCSK9 Variants

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serineprotease involved in regulating the levels of the low densitylipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah andPrat, 2007). In vitro experiments have shown that adding PCSK9 to HepG2cells lowers the levels of cell surface LDLR (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004).Experiments with mice have shown that increasing PCSK9 protein levelsdecreases levels of LDLR protein in the liver (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004), whilePCSK9 knockout mice have increased levels of LDLR in the liver (Rashidet al., 2005). Additionally, various human PCSK9 mutations that resultin either increased or decreased levels of plasma LDL have beenidentified (Kotowski et al., 2006; Zhao et al., 2006). PCSK9 has beenshown to directly interact with the LDLR protein, be endocytosed alongwith the LDLR, and co-immunofluoresce with the LDLR throughout theendosomal pathway (Lagace et al., 2006).

PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8)family of serine proteases (Seidah et al., 2003). Humans have nineprohormone-proprotein convertases that can be divided between the S8Aand S8B subfamilies (Rawlings et al., 2006). Furin, PC1/PC3, PC2, PACE4,PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily S8B.Crystal and NMR structures of different domains from mouse furin and PC1reveal subtilisin-like pro- and catalytic domains, and a P domaindirectly C-terminal to the catalytic domain (Henrich et al., 2003;Tangrea et al., 2002). Based on the amino acid sequence similaritywithin this subfamily, all seven members are predicted to have similarstructures (Henrich et al., 2005). SKI-1/SIP and PCSK9 are classified insubfamily S8A. Sequence comparisons with these proteins also suggest thepresence of subtilisin-like pro- and catalytic domains (Sakai et al.,1998; Seidah et al., 2003; Seidah et al., 1999). In these proteins theamino acid sequence C-terminal to the catalytic domain is more variableand does not suggest the presence of a P domain.

Prohormone-proprotein convertases are expressed as zymogens and theymature through a multi step process. The function of the pro-domain inthis process is two-fold. The pro-domain first acts as a chaperone andis required for proper folding of the catalytic domain (Ikemura et al.,1987). Once the catalytic domain is folded, autocatalysis occurs betweenthe pro-domain and catalytic domain. Following this initial cleavagereaction, the pro-domain remains bound to the catalytic domain where itthen acts as an inhibitor of catalytic activity (Fu et al., 2000). Whenconditions are correct, maturation proceeds with a second autocatalyticevent at a site within the pro-domain (Anderson et al., 1997). Afterthis second cleavage event occurs the pro-domain and catalytic domaindissociate, giving rise to an active protease.

Autocatalysis of the PCSK9 zymogen occurs between Gln152 and Ser153(VFAQ|SIP (SEQ ID NO: 67)) (Naureckiene et al., 2003), and has beenshown to be required for its secretion from cells (Seidah et al., 2003).A second autocatalytic event at a site within PCSK9's pro-domain has notbeen observed. Purified PCSK9 is made up of two species that can beseparated by non-reducing SDS-PAGE; the pro-domain at 17 Kd, and thecatalytic plus C-terminal domains at 65 Kd. PCSK9 has not been isolatedwithout its inhibitory pro-domain, and measurements of PCSK9's catalyticactivity have been variable (Naureckiene et al., 2003; Seidah et al.,2003).

In certain embodiments, a PCSK9 polypeptide includes terminal residues,such as, but not limited to, leader sequence residues, targetingresidues, amino terminal methionine residues, lysine residues, tagresidues and/or fusion protein residues. “PCSK9” has also been referredto as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin/kexin type9, and neural apoptosis regulated convertase 1. The PCSK9 gene encodes aproprotein convertase protein that belongs to the proteinase K subfamilyof the secretory subtilase family. The term “PCSK9” denotes both theproprotein and the product generated following autocatalysis of theproprotein. When only the autocatalyzed product is being referred to(such as for an antigen binding protein or ligand that binds to thecleaved PCSK9), the protein can be referred to as the “mature,”“cleaved”, “processed” or “active” PCSK9. When only the inactive form isbeing referred to, the protein can be referred to as the “inactive”,“pro-form”, or “unprocessed” form of PCSK9. The term PCSK9 alsoencompasses PCSK9 molecules incorporating post-translationalmodifications of the PCSK9 amino acid sequence, such as PCSK9 sequencesthat have been glycosylated, PCSK9 sequences from which its signalsequence has been cleaved, PCSK9 sequence from which its pro domain hasbeen cleaved from the catalytic domain but not separated from thecatalytic domain (see, e.g., FIGS. 1A and 1B of US20120093818A1; whichis incorporated by reference herein in its entirety).

The present invention provides anti-PCSK9 ligands; and PCSK9-binding ortargeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of PCSK9, in particular human PCSK9 or its ligands and inscreening assays to identify other antagonists of PCSK9 activity. Someof the ligands of the invention are useful for inhibiting binding ofPCSK9 to LDLR, or inhibiting PCSK9-mediated activities.

Anti-PCSK9 ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human PCSK9, which is a commonly-occurring form (see, eg,US20120093818A1 and US20110065902A1; each of which is incorporated byreference herein in its entirety). While such therapies are useful forhuman patients harbouring this form of human PCSK9, the inventorconsidered it useful to investigate the possibility of targeting muchrarer—but still naturally-occurring—forms of PCSK9 amongst humanpopulations. In this way, the inventor arrived at insight into thenatural occurrences and distributions of rarer human PCSK9 forms thatcan serve as useful targets (at the protein or nucleic acid level) forhuman treatment, prophylaxis and diagnosis pertinent to diseases andconditions mediated or associated with PCSK9 activity. This particularlyprovides for tailored therapies, prophylaxis and diagnosis in humansthat are devoid of the common PCSK9 gene or protein (ie, the form a ora′ as used in US20120093818A1 and US20110065902A1 to generateantibodies).

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer PCSK9 polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), in many of the examples determined by the inventor,comprise multiple changes at the nucleotide and amino acid levels fromthe corresponding common form nucleotide and amino acids sequences, ie,there are multiple non-synonymous changes at the nucleotide level thattranslate into multiple corresponding changes in the protein target inhumans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor realised that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-PCSK9 ligand for administration to human patients fortherapy and/or prophylaxis of PCSK9-mediated or associated diseases orconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, the present inventor decided to determine aset of human PCSK9 variants on the basis of the following criteria,these being criteria that the inventor realised would provide for usefulmedical drugs and diagnostics to tailored need in the human population.The inventor selected variants having at least 3 of the 4 followingcriteria:—

-   -   PCSK9 variants having a cumulative human allele frequency in the        range from 1 to 10%;    -   PCSK9 variants having a total human genotype frequency in the        range from 1 to about 15%;    -   PCSK9 variants found in many different human ethnic populations        (using the standard categorisation of the 1000 Genomes Project,        which is an accepted standard in the art; see Table 4 below);        and    -   PCSK9 variants found in many individuals distributed across such        many different ethnic populations.

On the basis of these criteria, the inventor identified the variantslisted in Table 1 below (excluding form a).

The inventor's selection included, as a consideration, selection fornucleotide variation that produced amino acid variation in correspondingPCSK9 forms (ie, non-synonymous variations), as opposed to silentvariations that do not alter amino acid residues in the target protein.

TABLE 1 Human PCSK9 variants distributed over several human ethnicpopulations & having a total human genotype frequency in the range of 1to about 15% (a) Amino acid variability, population distributions andfrequencies Form 46R 53A 425N 443A 4741 619Q 670E ASW, 939 14 0.39510.4506 0.64815 α YRI, (0.8457) GBR, TSI, CLM, CHB, LWK, CHS, MXL, JPT,PUR, IBS, FIN, CEU Var- Amino Acid Position & Variation Human No. No.Hom Freq⁴ iant Popu- Indiv- Unique Het (Het + Cum Form 46L 53V 425S 443T474V 619P 670G lations ids¹ Pops² Freq³ Hom freq⁵) Freq⁶ f x ASW, 180 120.153 0.009 0.0855 YRI, (0.162) GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS,FIN, CEU c x ASW, 153 12 0.1296 0.0081 0.0729 YRI, (0.1377) GBR, TSI,CLM, CHB, LWK, CHS, JPT, PUR, FIN, CEU r x x 0.0234 0.009 0.0292(0.0324) p x x ASW,  49  9 0.0441 (0.0441) 0.0221 GBR,     TSI,     CLM,  JPT,   PUR,   IBS,   FIN,   CEU   m x LWK,  29  4 0.0225 (0.0225)0.0149 ASW,     YRI,   CLM   e x x LWK,  15  3 0.0135 (0.0135) 0.0068ASW,   YRI   h x x LWK,  10  3 0.009 (0.009) 0.0045 ASW,   YRI   aj X xPUR,   9  4 0.0081 (0.0081) 0.0041 TSI,      FIN,    CEU    q x x CHS,  7  5 0.0063 (0.0063) 0.0032 ASW, JPT, PUR, CHB (b) Nucleotide SequenceVariations of Selected Alleles G C A G A A A Nucleotide Position¹ 1: 1:1: 1: 1: 1: 1: 55505647 55505668 55523802 55523855 55524237 5552722255529187 Non-Synonymous Nucleotide Variation² T T G A G C G All- VariantID³ ele rs rs rs rs rs rs rs α 11591147 11583680 28362261 28362263562556 28362277 505151 Var- iant All- Corresponding Amino Acid Variationele 46L 53V 425S 443T 474V 619P 670G f X c X r X X p X X m X e X X h X Xaj X X q X X “x” in a box indicates that the amino acid for the variantform is different from the amino acid at that position in form α, thevariant amino acid being shown in “Amino Acid Position & Variation” ofthe table and the form α amino acid being shown in the first row of thetable; amino acids at all other positions of each variant form areidentical to those found in form α. “x” in a box indicates that avariant allele comprises the non-synonymous nucleotide variationindicated in the 5th row. ¹Notation is chromosome number (all positionsare on human chromosome 1): coordinate number (Ensembl release73-September 2013, Genome assembly: GRCh37 (GCA_000001405.13);²Nucleotide change (compared to allele a nucleotide shown in first row)giving rise to an amino acid change in the variant form (compared toamino acid of allele α); and ³NCBI dbSNP reference number (NCBI dbSNPBuild 138 released on Apr 25, 2013).Amino acid numbering is per the numbering shown for the pro-form inTable 2 below.1. Number of individuals in 1000 Genomes database found to have theallele;2. Number of unique human ethnic populations in 1000 Genomes database inwhich the allele was found to occur;3. Heterozygous human genotype frequency, ie, cumulative frequency ofall genotypes having one occurrence of the variant allele and oneoccurrence of another allele (heterozygous state), eg, ac genotype in1000 Genomes database;4. Homozygous human genotype frequency, ie, cumulative frequency of twooccurrences of the variant allele (homozygous state), eg, cc genotype in1000 Genomes database; and5. Total human genotype frequency, ie, total of heterozygous plushomozygous human genotype frequencies.6. Cumulative human allele frequency of all occurrences of the variantallele in 1000 Genomes database.Form a′ is identical to form a with the exception that form a′ has aglycine (G) at position 620 (see US20120093818 (Amgen, Inc)); form a hasE at this position.

TABLE 2 Sequences(a) Human PCSK9 Form a Amino Acid Sequence (SEQ ID NO: 1) - “Pro-form”with Signal Sequence                                             46     53MGRTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQPARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425              443                            474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                           619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQItalics = signal sequence 1-30 Courier = pro peptide 31-152 lower case =catalytic domain 153-449 UPPER CASE = C-terminal domain 450-692Underlined =residues changed from allele a in other sequences (aa residue number shown)The pro-form is the sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1.The mature form is the sequence from amino acid number 153 to (and including) amino acid number 692 of SEQ ID NO: 1.(b) Human PCSK9 Form a Amino Acid Sequence (SEQ ID NO: 3) -“Mature-form”(Numbering and notation as per SEQ ID NO: 1 above has been retained)sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425              443                            474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                           619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ(c) Human PCSK9 Allele a Nucleotide Sequence (SEQ ID NO: 28) - Encoding“Pro-form” Plus Signal SequenceATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC                                                      R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG            A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                       N425S AAT to AGT                                       A443T GCC to ACCatccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC    I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC Italics =nucleotide sequence encoding signal sequence (nucleotides 1-90)Courier = nucleotide sequence encoding pro peptide (nucleotides 91-456)lower case =nucleotide sequence encoding catalytic domain (nucleotides 457-1346)UPPER CASE =nucleotide sequence encoding C-terminal domain (nucleotides 1347-2076)Underlined = allelic variations from allele a in other sequences   (aa residue number changes and codon changes shown)The pro-form is encoded by nucleotide sequence from nucleotide 91 to (and including) nucleotide 2076.The mature form is encoded by nucleotide sequence from nucleotide 457 to (and including) nucleotide 2076.

Variant Allele Nucleotide Sequences

Thus,

(i) The nucleotide sequence of allele f is identical to SEQ ID NO: 28except that the nucleotide sequence of allele f comprises a GTC codoninstead of an ATC codon at the position labelled “I474V” in SEQ ID NO:28;(ii) The nucleotide sequence of allele c is identical to SEQ ID NO: 28except that the nucleotide sequence of allele c comprises a GGG codoninstead of an GAG codon at the position labelled “E670G” in SEQ ID NO:28;(iii) The nucleotide sequence of allele r is identical to SEQ ID NO: 28except that the nucleotide sequence of allele r comprises a GTC codoninstead of an ATC codon at the position labelled “I474V” in SEQ ID NO:28; and a GGG codon instead of an GAG codon at the position labelled“E670G” in SEQ ID NO: 28;(iv) The nucleotide sequence of allele p is identical to SEQ ID NO: 28except that the nucleotide sequence of allele p comprises a GTC codoninstead of a GCC codon at the position labelled “A53V” in SEQ ID NO: 28;and a GTC codon instead of an ATC codon at the position labelled “I474V”in SEQ ID NO: 28;(v) The nucleotide sequence of allele m is identical to SEQ ID NO: 28except that the nucleotide sequence of allele m comprises a ACC codoninstead of a GCC codon at the position labelled “A443T” in SEQ ID NO:28;(vi) The nucleotide sequence of allele e is identical to SEQ ID NO: 28except that the nucleotide sequence of allele e comprises a AGT codoninstead of an AAT codon at the position labelled “N425S” in SEQ ID NO:28; and a GTC codon instead of an ATC codon at the position labelled“I474V” in SEQ ID NO: 28;(vii) The nucleotide sequence of allele h is identical to SEQ ID NO: 28except that the nucleotide sequence of allele h comprises a ACC codoninstead of a GCC codon at the position labelled “A443T” in SEQ ID NO:28; and a CCG codon instead of a CAG codon at the position labelled“Q619P” in SEQ ID NO: 28;(viii) The nucleotide sequence of allele aj is identical to SEQ ID NO:28 except that the nucleotide sequence of allele aj comprises a CTTcodon instead of an CGT codon at the position labelled “R46L” in SEQ IDNO: 28; and a GTC codon instead of an ATC codon at the position labelled“I474V” in SEQ ID NO: 28; and(ix) The nucleotide sequence of allele q is identical to SEQ ID NO: 28except that the nucleotide sequence of allele q comprises a GTC codoninstead of a GCC codon at the position labelled “A53V” in SEQ ID NO: 28;and a GGG codon instead of an GAG codon at the position labelled “E670G”in SEQ ID NO: 28.

Variant Pro-Form Amino Acid Sequences

(Numbering is as per SEQ ID NO: 1 recited above)(A) The amino acid sequence of form f is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form fcomprises a valine at position 474;(B) The amino acid sequence of form c is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ccomprises a glycine at position 670;(C) The amino acid sequence of form r is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form rcomprises a valine at position 474 and a glycine at position 670;(D) The amino acid sequence of form p is identical the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form pcomprises a valine at position 53 and a valine at position 474;(E) The amino acid sequence of form m is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form mcomprises a threonine at position 443;(F) The amino acid sequence of form e is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ecomprises a serine at position 425 and a valine at position 474;(G) The amino acid sequence of form h is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form hcomprises a threonine at position 443 and a proline at position 619;(H) The amino acid sequence of form aj is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ajcomprises a leucine at position 46 and a valine at position 474; and(I) The amino acid sequence of form q is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form qcomprises a valine at position 53 and a glycine at position 670.

Variant Mature Form Amino Acid Sequences

(Numbering is as per SEQ ID NO: 1 recited above)(A′) The amino acid sequence of form f is identical to SEQ ID NO: 2except that the amino acid sequence of form f comprises a valine atposition 474;(B′) The amino acid sequence of form c is identical to SEQ ID NO: 2except that the amino acid sequence of form c comprises a glycine atposition 670;(C′) The amino acid sequence of form r is identical to SEQ ID NO: 2except that the amino acid sequence of form r comprises a valine atposition 474 and a glycine at position 670;(D′) The amino acid sequence of form p is identical to SEQ ID NO: 2except that the amino acid sequence of form p comprises a valine atposition 474;(E′) The amino acid sequence of form m is identical to SEQ ID NO: 2except that the amino acid sequence of form m comprises a threonine atposition 443;(F′) The amino acid sequence of form e is identical to SEQ ID NO: 2except that the amino acid sequence of form e comprises a serine atposition 425 and a valine at position 474;(G′) The amino acid sequence of form h is identical to SEQ ID NO: 2except that the amino acid sequence of form h comprises a threonine atposition 443 and a proline at position 619;(H′) The amino acid sequence of form aj is identical to SEQ ID NO: 2except that the amino acid sequence of form aj comprises valine atposition 474; and(I′) The amino acid sequence of form q is identical to SEQ ID NO: 2except that the amino acid sequence of form q comprises a glycine atposition 670.

The mature form of p is identical to the mature form off and aj.

The mature form of c is identical to the mature form of q.

Further sequence analysis and 3D in silico modelling (see FIG. 1)revealed that selected variants also fulfilled the following selectioncriteria:—

-   -   PCSK9 variants whose variant amino acid residues (versus the        common form of human PCSK9) are found in the mature form of the        target (ie, outside the pro-domain); and    -   PCSK9 variants whose variant amino acid residues (versus the        common form of human PCSK9) are surface-exposed on the target,        which the inventor saw as contributing to determining the        topography of the target and potentially contributing to how and        where ligand binding on the target occurs.

As shown in FIG. 1, identified positions 425, 443, 474, 619 and 670(found in the selected variants of the invention) are allsurface-exposed and outside of the pro-domain. Variant positions 425 and443 are surface-exposed on the catalytic domain, while variant positions474, 619 and 670 are surface-exposed on the C-terminal domain.

In a first example, the invention addresses the need to treat humanshaving naturally-occurring rarer natural PCSK9 alleles, genotypes andphenotypes (rarer protein forms). In this respect, the inventionprovides the following aspects:

In a First Aspect:

An anti-human PCSK9 ligand for use in a method of treating and/orpreventing a PCSK9-mediated disease or condition in a human whose genomecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 29-37, wherein the method comprises administering the ligandto the human.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Second Aspect:

The ligand of aspect 1, wherein the ligand has been or is determined ascapable of binding a human PCSK9 selected from the group consistingforms f, c, r, p, m, e, h, aj and q.

In an example of any aspect, the ligand binds (or has been determined tobind) two, three, four or more human PCSK9 selected from the groupconsisting forms f, c, r, p, m, e, h, aj and q.

In an example of any aspect, the ligand comprises a protein domain thatspecifically binds to PCSK9, eg, a human PCSK9 selected from the groupconsisting forms f, c, r, p, m, e, h, aj and q.

The term “specifically binds,” or the like, means that a ligand, eg, anantibody or antigen-binding fragment thereof, forms a complex with anantigen that is relatively stable under physiologic conditions. Specificbinding can be characterized by an equilibrium dissociation constant ofat least about 1×10⁻⁶M or less (e.g., a smaller KD denotes a tighterbinding). Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds a human PCSK9 may, however, exhibitcross-reactivity to other antigens such as a PCSK9 molecule from anotherspecie. Moreover, multi-specific antibodies (e.g., bispecifics) thatbind to human PCSK9 and one or more additional antigens are nonethelessconsidered antibodies that “specifically bind” PCSK9, as used herein.

In an example of any aspect, the ligand comprises or consists of aprotein that mimics the EGFA domain of the LDL receptor and specificallybinds to PCSK9, eg, a human PCSK9 selected from the group consistingforms f, c, r, p, m, e, h, aj and q.

In an example of any aspect, the ligand antagonises PCSK9, eg, a humanPCSK9 selected from the group consisting forms f, c, r, p, m, e, h, ajand q.

In an example of any aspect, the method comprises (before administeringthe ligand) the step of determining that the ligand is capable ofbinding a human PCSK9 selected from the group consisting forms f, c, r,p, m, e, h, aj and q.

In an example of any aspect, binding is determined by SPR. In an exampleof any aspect, binding is determined by ELISA.

In an example of any aspect, said forms are the mature forms.

In an example of any aspect, said forms are the pro-forms.

In a Third Aspect:

A ligand that binds a human PCSK9 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4-27 for use in amethod comprising the step of using the ligand to target said PCSK9 in ahuman to treat and/or prevent a disease or condition mediated by PCSK9,the method comprising administering the ligand to the human.

In an example, the disease or condition is mediated by a human PCSK9comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the groupconsisting of SEQ ID NOs: 4-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These arenaturally-occurring sequences that do not comprise 46L and which meetthe criteria set out above. These groups comprise variants that areassociated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, thatcomprises a 425S, which is associated with elevated LDL-C (Pisciotta etal 2006).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670Gwhich is a marker for severity of coronary atherosclerosis (Chen et al2005).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequencesthat have a naturally-occurring combination of differences from SEQ IDNOs: 1-3 (form a) and which meet the criteria set out above.

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Fourth Aspect:

The ligand of aspect 3, wherein the genome of the human comprises anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33

In an example, the nucleotide sequence is SEQ ID NO: 34

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Fifth Aspect:

The ligand of any preceding aspect, wherein the human has been or isgenotyped as positive for a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Sixth Aspect:

The ligand of any preceding aspect, wherein the human has been or isphenotyped as positive for a human PCSK9 selected from the groupconsisting of forms f, c, r, p, m, e, h, aj and q or at least thecatalytic or C-terminal domain thereof

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Seventh Aspect:

The ligand of any preceding aspect, wherein the method comprisesgenotyping the human as positive for a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eighth Aspect:

The ligand of any preceding aspect, wherein the method comprisesphenotyping the human has positive for a human PCSK9 selected from thegroup consisting of forms f c, r, p, m, e, h, aj and q or at least thecatalytic or C-terminal domain thereof.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Ninth Aspect:

The ligand of any preceding aspect, wherein the human has been or isgenotyped as heterozygous for a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof optionally wherein thehuman has been or is genotyped as comprising the nucleotide sequence ofSEQ ID NO: 28 or at least the catalytic domain- or C-terminaldomain-encoding sequence thereof and a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof.

“Heterozygous” here means that in the human's genotype one allelecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminaldomain-encoding sequence thereof and other allele can be any PCSK9 (eg,form a, a′ or an allele comprising a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof).

In an example, the method comprises (before administering the ligand)genotyping the human as heterozygous for a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof; optionally alsogenotyping the human as comprising the nucleotide sequence of SEQ ID NO:28 or at least the catalytic domain- or C-terminal domain-encodingsequence thereof and a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37.

These are naturally-occurring allele (haplotype) sequences that do notencode 46L and which meet the criteria set out above. These groupscomprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Tenth Aspect:

The ligand of any one of aspects 1 to 9, wherein the genome of the humanhas been or is genotyped as homozygous for a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof

“Homozygous” here means that in the human's genotype each allelecomprises the same nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof.

In an example, the method comprises genotyping the human as homozygousfor a nucleotide sequence selected from the group consisting of SEQ IDNOs: 29-37 or at least the catalytic domain- or C-terminaldomain-encoding sequence thereof.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eleventh Aspect:

The ligand of any preceding aspect, wherein the ligand comprises anantibody binding site that binds a human PCSK9 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 4-27 andoptionally has been or is determined as capable of such binding.

In an example, the method comprises (before administering the ligand)the step of determining that the ligand is capable of binding to saidhuman PCSK9.

In an example, the binding is specific binding. In an example, theligand binds (or has been determined as binding) to the PCSK9 with anaffinity (Kd) of 1 mM, 100 nM, 10 nM or 1 nM or less. In an embodiment,the affinity is no less than 10, 100 or 1000 fM.

In an example, binding or affinity is determined by SPR or ELISA.

In an example, the disease or condition is mediated by a human PCSK9comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the groupconsisting of SEQ ID NOs: 4-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These arenaturally-occurring sequences that do not comprise 46L and which meetthe criteria set out above. These groups comprise variants that areassociated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, thatcomprises a 425S, which is associated with elevated LDL-C (Pisciotta etal 2006).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670Gwhich is a marker for severity of coronary atherosclerosis (Chen et al2005).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequencesthat have a naturally-occurring combination of differences from SEQ IDNOs: 1-3 (form a) and which meet the criteria set out above

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Twelfth Aspect:

The ligand of aspect 11, wherein the ligand is an antibody or antibodyfragment. For example, the antibody or antibody fragment is a PCSK9antagonist, eg, neutralises PCSK9.

Examples of such antibodies are disclosed, for instance, in WO2008/057457, WO2008/057458, WO 2008/057459, WO 2008/063382, WO2008/133647, WO 2009/100297, WO 2009/100318, WO 2011/037791, WO2011/053759, WO 2011/053783, WO 2008/125623, WO 2011/072263, WO2009/055783, WO 2010/029513, WO 2011/11 1007, WO 2010/077854, thedisclosures and sequences of such antibodies being incorporated hereinfor use in the invention in their entireties by reference. One specificexample is AMG 145 (Amgen), LY3015014 (Eli Lilly) or alirocumab.Advantageously, the ligand is or comprises alirocumab. Alternatively,the ligand is or comprises evolocumab.

In an example, the ligand is SAR236553/REGN727 (SanofiAventis/Regeneron) or a PCSK9-binding derivative thereof.

In an example, the ligand comprises or consists of a neutralizingantibody that binds to the PCSK9, wherein the antibody binds to PCSK9and reduces the likelihood that PCSK9 binds to LDLR.

The ligand of aspect 11, wherein the ligand is a PCSK9 antagonist, eg,neutralises PCSK9.

In an example of any aspect of the invention, the ligand comprises orconsists a ligand selected from evolocumab, 1D05-IgG2 (Merck & Co.),ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat), LY3015014 (Eli Lilly) andalirocumab (SAR236553/REGN727; Sanofi Aventis/Regeneron).

In Thirteenth Aspect: The ligand of any one of aspects 1 to 10, wherein(i) the ligand comprises a sequence of contiguous nucleotides thatspecifically hybridises to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof, or specifically hybridisesto an antisense sequence or an RNA transcript of said sequence, whereinsaid sequence of contiguous nucleotides hybridises to at least onenucleotide present in said selected sequence which is not present in SEQID NO: 28 or hybridises to an antisense sequence or an RNA transcriptthereof respectively; and/or (ii) the ligand comprises a sequence of atleast 10 contiguous nucleotides of a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or is an antisense sequence orRNA version of said contiguous nucleotides, wherein said sequence ofcontiguous nucleotides comprises at least one nucleotide present in saidselected sequence which is not present in SEQ ID NO: 28.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an embodiment, the ligand comprises at least 10, 11, 12, 13, 14, 15,20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides of saidnucleotide sequence.

In a Fourteenth Aspect:

The ligand of any preceding aspect, wherein said disease or condition ishyperlipidaemia, hypercholesterolaemia (eg, familialhypercholesterolaemia), heart attack, stroke, coronary heart disease,atherosclerosis or a cardiovascular disease or condition.

The ligand of any preceding aspect, wherein the disease or condition ishypercholesterolemia, hyperlipidemia, hypercholesterolemia,dyslipidemia, cholestatic liver disease, nephrotic syndrome,hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In an example, said disease or condition is hypercholesterolaemia. Theterm “hypercholesterolaemia,” as used herein, refers to a condition inwhich cholesterol levels are elevated above a desired level. In someembodiments, this denotes that serum cholesterol levels are elevated. Insome embodiments, the desired level takes into account various “riskfactors” that are known to one of skill in the art (and are described orreferenced in US20120093818).

The ligand of any preceding aspect, wherein the human is identified asheterozygous for Familial Hypercholesterolemia, statin intolerant,statin uncontrolled, or at risk for developing hypercholesterolemia,dyslipidemia, cholestatic liver disease, nephrotic syndrome,hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In a Fifteenth Aspect:

The ligand of any preceding aspect, wherein said disease or condition isassociated with elevated LDL cholesterol.

Cholesterol levels are measured in milligrams (mg) of cholesterol perdeciliter (dL) of blood in the United States and some other countries.Canada and most European countries measure cholesterol in millimoles(mmol) per liter (L) of blood. Below are general guideline ideal rangesand elevated ranges.

Total cholesterol Total cholesterol* (U.S. and some (Canada and mostother countries) of Europe) Below 200 mg/dL Below 5.2 mmol/L Ideal200-239 mg/dL 5.2-6.2 mmol/L Borderline high 240 mg/dL and above Above6.2 mmol/L High LDL cholesterol LDL cholesterol* (U.S. and some (Canadaand most other countries) of Europe) 100-129 mg/dL 2.6-3.3 mmol/L Ideal130-159 mg/dL 3.4-4.1 mmol/L Borderline high 160-189 mg/dL 4.1-4.9mmol/L High 190 mg/dL and above Above 4.9 mmol/L Very high *Canadian andEuropean guidelines differ slightly from U.S. guidelines. Theseconversions are based on U.S. guidelines.

Elevated LDL cholesterol is, therefore, 160 mg/dL or above (4.1 mmol/Lor above).

In a Sixteenth Aspect:

The ligand of any preceding aspect, wherein the ligand inhibits humanPCSK9 binding to human LDL receptor and optionally has been or isdetermined as capable of such inhibition.

In an example, the method comprises (before administering the ligand)determining that the ligand is capable of such inhibition.

Inhibition determination is eg, inhibition in a blood or serum sample,at rtp, at ph7, at 37 degrees centigrade and/or under the physiologicalconditions of a human body.

In a Seventeenth Aspect:

The ligand of any preceding aspect, wherein the human is resistant orsubstantially resistant to statin (eg, avorstatin and/or fluvastatin)treatment of said disease or condition.

In an Eighteenth Aspect:

The ligand of any preceding aspect, wherein the ligand is for treatingand/or preventing a PCSK9-mediated disease or condition in a human

(i) whose genome comprises SEQ ID NO: 29 and wherein the human is ofASW, YRI, GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry;or(ii) whose genome comprises SEQ ID NO: 30 and wherein the human is ofASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry;or(iii) whose genome comprises SEQ ID NO: 32 and wherein the human is ofASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or(iv) whose genome comprises SEQ ID NO: 33 and wherein the human is ofLWK, ASW, YRI or CLM ancestry; or(v) whose genome comprises SEQ ID NO: 34 and wherein the human is ofLWK, ASW or YRI ancestry; or(vi) whose genome comprises SEQ ID NO: 35 and wherein the human is ofPUR, TSI, FIN or CEU ancestry; or(vii) whose genome comprises SEQ ID NO: 36 and wherein the human is ofLWK, ASW or YRI ancestry; or(viii) whose genome comprises SEQ ID NO: 37 and wherein the human is ofCHS, ASW, JPT, PUR or CHB ancestry.

In a Ninteenth Aspect:

The ligand of any preceding aspect, wherein the ligand is for treatingand/or preventing a PCSK9-mediated disease or condition in a human

(i) that expresses PCSK9 form f and wherein the human is of ASW, YRI,GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry; or(ii) that expresses PCSK9 form c and wherein the human is of ASW, YRI,GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry; or(iii) that expresses PCSK9 form p and wherein the human is of ASW, GBR,TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or(iv) that expresses PCSK9 form m and wherein the human is of LWK, ASW,YRI or CLM ancestry; or(v) that expresses PCSK9 form e and wherein the human is of LWK, ASW orYRI ancestry; or(vi) that expresses PCSK9 form h and wherein the human is of PUR, TSI,FIN or CEU ancestry; or(vii) that expresses PCSK9 form aj and wherein the human is of LWK, ASWor YRI ancestry; or(viii) that expresses PCSK9 form q and wherein the human is of CHS, ASW,JPT, PUR or CHB ancestry.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twentieth Aspect:

A pharmaceutical composition or kit for treating and/or preventing aPCSK9-mediated condition or disease (eg, as recited in aspect 14 or 15),the composition or kit comprising a ligand of any preceding aspect andoptionally a statin (eg, cerovastatin, atorvastatin, simvastatin,pitavastin, rosuvastatin, fluvastatin, lovastatin or pravastatin); andoptionally in combination with a label or instructions for use to treatand/or prevent said disease or condition in a human (eg, coveringtreatment of a human as recited in aspect 18 or 19); optionally whereinthe label or instructions comprise a marketing authorisation number (eg,an FDA or EMA authorisation number); optionally wherein the label orinstructions comprise directions to administer alirocumab or evolocumabto said human; optionally wherein the kit comprises an IV or injectiondevice that comprises the ligand (and, eg, also a statin).

In a Twenty-First Aspect:

A method of producing an anti-human PCSK9 antibody binding site, themethod comprising obtaining a plurality of anti-PCSK9 antibody bindingsites, screening the antibody binding sites for binding to a human PCSK9selected from the group consisting of forms f, c, r, p, m, e, h, aj andq or a catalytic or C-terminal domain or a peptide thereof thatcomprises amino acid variation from the corresponding sequence of SEQ IDNO: 1, 2 or 3 and isolating an antibody binding site that binds in thescreening step, and optionally producing a form f c, r, p, m, e, h, ajor q PCSK9-binding fragment or derivative of the isolated antibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example of this and the next aspect, the plurality of bindingsites comprises or consists of a plurality of 4-chain antibodies orfragments thereof, eg, dAbs, Fabs or scFvs. Suitable methods forproducing pluralities of binding sites for screening include phagedisplay (producing a phage display library of antibody binding sites),ribosome display (producing a ribosome display library of antibodybinding sites), yeast display (producing a yeast display library ofantibody binding sites), or immunisation of a non-human vertebrate (eg,a rodent, eg, a mouse or rat, eg, a Velocimouse™, Kymouse™, Xenomouse™,Aliva Mouse™, HuMab Mouse™, Omnimouse™, Omnirat™ or MeMo Mouse™) with aPCSK9 epitope and isolation of a repertoire of antibody-producing cells(eg, a B-cell, plasma cell or plasmablast repertoire) and/or arepertoire of isolated antibodies.

In an example, the method comprises selecting one or more antibodybinding sites that each specifically binds to a human PCSK9 epitopecomprising amino acid variation from the corresponding sequence of SEQID NO: 1, 2 or 3.

In a Twenty-Second Aspect:

A method of producing an anti-human PCSK9 antibody, the methodcomprising immunising a non-human vertebrate (eg, a mouse or a rat) witha human PCSK9 comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences of forms f, c, r, p, m, e, h, ajand q or a catalytic or C-terminal domain or a peptide thereof thatcomprises amino acid variation from the corresponding sequence of SEQ IDNO: 1, 2 or 3 and isolating an antibody that binds a human PCSK9comprising selected from the group consisting of forms f, c, r, p, m, e,h, aj and q or a catalytic or C-terminal domain or a peptide thereofthat comprises amino acid variation from the corresponding sequence ofSEQ ID NO: 1, 2 or 3, and optionally producing a form f, c, r, p, m, e,h, aj or q PCSK9-binding fragment or derivative of the isolatedantibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-Third Aspect:

The method of aspect 21 or 22, comprising the step of obtaining anucleic acid encoding the antibody, fragment, derivative or binding siteand optionally inserting the nucleic acid in an expression vector.

For example, the method comprises isolating a cell (eg, B-cell,plasmablast, plasma cell or memory cell) comprising the nucleic acid,wherein the cell is obtained from a non-human vertebrate that has beenimmunised with the PCSK9 epitope.

In a Twenty-Fourth Aspect:

A kit for PCSK9 genotyping a human, wherein the kit comprises a nucleicacid (i) comprising a sequence of 10 or more (eg, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100 or more) contiguous nucleotides thatspecifically hybridises to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof, or specifically hybridisesto an antisense sequence or an RNA transcript of said sequence, whereinsaid sequence of contiguous nucleotides hybridises to at least onenucleotide present in said selected sequence which is not present in SEQID NO: 28 or hybridises to an antisense sequence or an RNA transcriptthereof; and/or (ii) comprising a sequence of at least 10 or more (eg,10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) nucleotides of anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or comprising an antisense sequence or RNA version of saidcontiguous nucleotides, wherein said sequence of contiguous nucleotidescomprises at least one nucleotide present in said selected sequencewhich is not present in SEQ ID NO: 28.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Twenty-Fifth Aspect:

A kit for PCSK9 genotyping or phenotyping a human, wherein the kitcomprises a ligand according to any one of aspects 1 to 19 or anantibody, fragment or derivative produced by the method of any one ofaspects 21 to 23.

In a Twenty-Sixth Aspect:

Use of an anti-PCSK9 ligand that binds a human PCSK9 selected from thegroup consisting of forms f, c, r, p, m, e, h, aj and q in themanufacture of a medicament for treating and/or preventing aPCSK9-mediated disease or condition in a human whose genome comprises anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37, optionally for treating and/or preventing a PCSK9-mediateddisease or condition in a human as recited in aspect 18 or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-Seventh Aspect:

Use of an anti-PCSK9 ligand that binds a human PCSK9 selected from thegroup consisting of forms f, c, r, p, m, e, h, aj and q in themanufacture of a medicament for targeting said PCSK9 in a human to treatand/or prevent a disease or condition mediated by PCSK9, optionally fortargeting PCSK9 in a human as recited in aspect 18 or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Twenty-Eight Aspect:

The use of aspect 26 or 27, wherein the ligand, human, disease orcondition is according to any one of aspects 1 to 19.

In a Twenty-Ninth Aspect:

A method of targeting a PCSK9 for treating and/or preventing aPCSK9-mediated disease or condition in a human, the method comprisingadministering an anti-PCSK9 ligand to a human comprising a nucleotidesequence selected from the group consisting SEQ ID NOs: 29-37, whereby aPCSK9 encoded by said nucleotide sequence is targeted.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirtieth Aspect:

The method of aspect 29, wherein the method comprises targeting a humanPCSK9 selected from the group consisting of forms f, c, r, p, m, e, h,aj and q with said ligand to treat and/or prevent said disease orcondition in said human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-First Aspect:

A method of treating and/or preventing a disease or condition mediatedby PCSK9 in a human, the method comprising targeting a human PCSK9selected from the group consisting of forms f, c, r, p, m, e, h, aj andq by administering to the human a ligand that binds said PCSK9 therebytreating and/or preventing said disease or condition in the human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirty-Second Aspect:

The method of aspect 31, wherein the genome of the human comprises anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Thirty-Third Aspect:

The method of any one of aspects 29 to 32, wherein the human has been oris genotyped as positive for a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminaldomain-encoding sequence thereof

In a Thirty-Fourth Aspect:

The method of any one of aspects 29 to 33, wherein the human has been oris phenotyped as positive for a human PCSK9 selected from the groupconsisting of forms f, c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-Fifth Aspect:

The method of any one of aspects 29 to 34, wherein the method comprisesgenotyping the human as positive for a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or the catalytic- orC-terminal domain-encoding sequence thereof

In a Thirty-Sixth Aspect:

The method of any one of aspects 29 to 35, wherein the method comprisesphenotyping the human as positive for a human PCSK9 sequence selectedfrom the group consisting of forms f, c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-Seventh Aspect:

The method of any one of aspects 29 to 36, wherein the human has been oris genotyped as heterozygous for a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminaldomain-encoding sequence thereof; optionally wherein the human has beenor is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28or the catalytic- or C-terminal domain-encoding sequence thereof and anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or the catalytic- or C-terminal domain-encoding sequence thereof

In a Thirty-Eighth Aspect:

The method of any one of aspects 29 to 37, wherein the genome of thehuman has been or is genotyped as homozygous for a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or thecatalytic- or C-terminal domain-encoding sequence thereof

In a Thirty-Ninth Aspect:

The method of any one of aspects 29 to 38, wherein the method comprisesgenotyping the human for a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminaldomain-encoding sequence thereof before administering the ligand to thehuman, wherein the ligand is determined to be capable of binding to aPCSK9 encoded by said selected sequence.

In a Fortieth Aspect:

The method of any one of aspects 29 to 39, wherein the ligand, human,disease or condition is according to any one of aspects 1 to 19.

In a Forty-First Aspect:

A method according to any one of aspects 29 to 40 for treating and/orpreventing a condition or disease as recited in aspect 14 or 15, themethod comprising administering said ligand and a statin (eg,cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin,fluvastatin, lovastatin or pravastatin) to the human.

In a Forty-Second Aspect:

The method of aspect 41, wherein the ligand and statin are administeredseparately.

In a Forty-Third Aspect:

The method of aspect 41, wherein the ligand and statin are administeredsimultaneously.

In a Forty-Fourth Aspect:

The method of any one of aspects 29 to 43, wherein the ligand isadministered by subcutaneous injection.

In a Forty-Fifth Aspect:

A method of PCSK9 genotyping a nucleic acid sample of a human, themethod comprising identifying in the sample the presence of a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or thecatalytic- or C-terminal domain-encoding sequence thereof.

In a Forty-Sixth Aspect:

A method of PCSK9 typing a protein sample of a human, the methodcomprising identifying in the sample the presence of a human PCSK9selected from the group consisting of forms f, c, r, p, m, e, h, aj andq.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Forty-Seventh Aspect:

The method of aspect 45 or 46, comprising obtaining a sample of serum,blood, faeces, hair, tissue, cells, urine or saliva from a human,whereby the nucleic acid or protein sample is obtained and used in thestep of identifying said sequence.

In a Forty-Eighth Aspect:

The method of any one of aspects 45 to 47, comprising using a ligandaccording to any one of aspects 1 to 19 to carry out said identifyingstep.

In a Forty-Ninth Aspect:

A method of treating and/or preventing in a human patient acardiovascular disease or condition, or a disease or condition that isassociated with elevated LDL cholesterol (eg, hypercholesterolaemia),wherein the patient is receiving or has previously received statintreatment for said disease or condition, the method comprising typingthe patient using a method of any one of aspects 45 to 48 andadministering a ligand according to one of aspects 1 to 19 whereby thehuman is treated or said disease or condition is prevented; optionallyalso reducing or stopping statin treatment.

In an example, said reducing or stopping comprises reducing the doseand/or dosing frequency of statin.

In a Fiftieth Aspect:

A diagnostic, therapeutic or prophylactic kit comprising a ligand thatis capable of binding to or has been or is determined as capable ofbinding to an amino acid sequence selected from SEQ ID NOs: 4-27 andinstructions for carrying out the method of any one of aspects 46 to 49and/or a label or instructions indicating or covering administration ofthe ligand to a human as defined in any one of aspects 1 to 19.

In a Fifty-First Aspect:

A diagnostic, therapeutic or prophylactic kit comprising a nucleic acidprobe comprising a nucleotide sequence that specifically hybridises to anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or an antisense sequence or RNA transcript thereof andinstructions for carrying out the method of aspect 45, 47 or 48.

In examples of the present invention, the ligand specifically binds tohuman PCSK9, eg, one or more of the rare PCSK9 variants disclosed herein(eg, one, two, three, more or all mature forms f, c, r, p, m, e, h, ajand q) and optionally also the a and/or a′ form. For example, the ligandspecifically binds to mature form f and/or c as well as form a.Determination of such binding can be performed by any antibody bindingtest as known in the art, eg, by surface plasmon resonance Binding toeach such form is, for example, respectively with a Kd of at least 1 mM,100 nM, 1 nM, 100 pM, 10 pM or 1 pM.

In an example, the ligand binds form a and a PCSK9 selected from thegroup consisting of forms f, c, r, p, m, e, h, aj and q, wherein theligand binding to said selected form is with a Kd (determined by SPR)that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.In an embodiment, both forms are mature forms. In an embodiment, bothforms are pro-forms.

In an example, the ligand binds form a and form f, wherein the ligandbinding to form f is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form c, wherein the ligandbinding to form c is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form r, wherein the ligandbinding to form r is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form p, wherein the ligandbinding to form p is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form m, wherein the ligandbinding to form m is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form e, wherein the ligandbinding to form e is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form h, wherein the ligandbinding to form h is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form aj, wherein the ligandbinding to form aj is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form q, wherein the ligandbinding to form q is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In examples of the present invention, the ligand neutralises humanPCSK9, eg, one or more of the rare PCSK9 variants disclosed herein (eg,one, two, three, more or all mature forms f, c, r, p, m, e, h, aj and q)and optionally also the a and/or a′ form. For example, the ligandneutralises mature form f and/or c as well as form a. Determination ofneutralisation can be performed, for example, by any neutralisationassay method disclosed in US20120093818A1 (Amgen, Inc) orUS20110065902A1 (Regeneron Pharmaceuticals, Inc). Ligands of theinvention that bind or target PCSK9 are useful, for example, fortherapeutic and prophylactic applications disclosed in US20120093818A1and US20110065902A1, these specific disclosures being incorporatedherein by reference for use in the present invention and for possibleinclusion in claims herein.

In embodiments where the ligand is used for therapeutic applications, anantigen binding protein can inhibit, interfere with or modulate one ormore biological activities of a PCSK9 (eg, one or more of the rarevariants disclosed herein and optionally also the a and/or a′ form). Inone embodiment, ligand binds specifically to human PCSK9 (eg, one ormore of the rare variants disclosed herein and optionally also the aand/or a′ form) and/or substantially inhibits binding of human PCSK9(eg, said one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form) to LDLR by at least 20%, eg,20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuringbinding in an in vitro competitive binding assay). In an example, theligand is an antibody.

In an embodiment, the ligand has a Kd of less (binding more tightly)than 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M for binding to one,two or more of the rare variants disclosed herein and optionally alsothe a and/or a′ form. In an example, Kd is determined using SPR.

In an embodiment, the ligand has an IC50 for blocking the binding ofLDLR to one or more of the rare PCSK9 variants disclosed herein (andoptionally also the a and/or a′ form) of less than 1 microM, 1000 nM to100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10pM, 10 pM to 1 pM.

In an embodiment, the ligand has an IC50 for blocking the binding ofLDLR to the a and/or a′ form of PCSK9 that is no more than 1000, 100,90, 80, 70, 60, 50, 40, 30, 20 or 10-fold more (ie, more inhibitory)than the IC50 for blocking the binding of LDLR to one or more of therare PCSK9 variants disclosed herein (eg, one or more PCSK9 proteinscomprising a sequence selected from SEQ ID NOs: 4 to 27). Additionallyor alternatively, for example, the ligand has an IC50 for blocking thebinding of LDLR to (i) the a and/or a′ form of less than 1 microM, 1000nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pMto 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pMto 10 pM, 10 pM to 1 pM, eg, in the range of 1 mM to 1 pM (eg, 1 mM to100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM) and (ii) oneor more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4to 27 of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nMto 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pMto 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, eg, in therange of 1 mM to 1 pM (eg, 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10pM; or 100 pM to 1 pM).

In an embodiment, the ligand binds to the a and/or a′ form of PCSK9 witha binding affinity (Kd) that is greater than up to 10%, greater than upto 20%, greater than up to 40%, greater than up to 50%, greater than upto 55%, greater than up to 60%, greater than up to 65%, greater than upto 70%, greater than up to 75%, greater than up to 80%, greater than upto 85%, greater than up to 90%, greater than up to 95% or greater thanup to 100% (ie, is double) relative to binding to a PCSK9 comprising asequence selected from SEQ ID NOs: 4 to 27. Such binding measurementscan be made using a variety of binding assays known in the art, eg,using surface plasmon resonance (SPR), such as by Biacore™ or using theProteOn XPR36™ (Bio-Rad®), or using KinExA® (Sapidyne Instruments, Inc).

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0.05% by volume, eg, in the presence of P20 (polysorbate20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the ligand which is an antibody isdetermined using SPR by

1. Coupling anti-mouse (or other relevant vertebrate) IgG (eg, BiacoreBR-1008-38) to a biosensor chip (eg, GLM chip) such as by primary aminecoupling;2. Exposing the anti-mouse IgG (vertebrate antibody) to a test IgGantibody to capture test antibody on the chip;3. Passing the test antigen over the chip's capture surface at 1024 nM,256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and4. And determining the affinity of binding of test antibody to testantigen using surface plasmon resonance, eg, under an SPR conditiondiscussed above (eg, at 25° C. in physiological buffer). SPR can becarried out using any standard SPR apparatus, such as by Biacore™ orusing the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, eg, using amodel inherent to the ProteOn XPR36™ analysis software.

In an embodiment, assaying or testing of a ligand of the invention iscarried out at or substantially at pH7 (eg, for in vitro tests andassays) and at or substantially at rtp.

One example of an IgG2 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 154, FIG. 3KK of US20120093818A1, which sequence isincorporated herein by reference.

One example of an IgG4 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 155, FIG. 3KK of US20120093818A1, which sequence isincorporated herein by reference.

One example of a kappa light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 157, FIG.3KK which sequence is incorporated herein by reference.

One example of a lambda light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 156, FIG.3KK of US20120093818A1, which sequence is incorporated herein byreference.

In examples of the present invention, the ligand binds mature PCSK9, eg,a mature form of one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the catalyticdomain of PCSK9, eg, of a mature form of one or more of the rarevariants disclosed herein and optionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the prodomain ofPCSK9, eg, of a mature form of one or more of the rare variantsdisclosed herein and optionally also the a and/or a′ form.

In some embodiments, the ligand binds to the V domain of PCSK9, eg, of amature form of one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form. In some embodiments, the ligandbinds to the V domain of PCSK9 (eg, of a mature form of one or more ofthe rare variants disclosed herein and optionally also the a and/or a′form) and prevents (or reduces, eg, by at least 10%) PCSK9 from bindingto LDLR. In some embodiments, the ligand binds to the V domain of PCSK9(eg, of a mature form of one or more of the rare variants disclosedherein and optionally also the a and/or a′ form), and while it does notprevent (or reduce) the binding of PCSK9 to LDLR, the ligand prevents orreduces (eg, by at least 10%) the adverse activities mediated throughPCSK9 on LDLR.

In examples of the present invention, the ligand is or comprises a fullyhuman antibody. In an example, the ligand comprises human variableregions or humanised variable regions.

In an example, the ligand of the invention specifically binds to anepitope of a human PCSK9 selected from the group consisting of forms f,c, r, p, m, e, h, aj and q, wherein the epitope comprises at least oneamino acid that is not found in form a. For example, the amino acid isselected from the group consisting of 46L, 53V, 425S, 443T, 474V, 619Pand 670G (numbering as used in SEQ ID NO:1). For example, the amino acidis selected from the group consisting of 425S, 443T, 474V, 619P and 670G(numbering as used in SEQ ID NO:1). For example, the amino acid isselected from the group consisting of 425S and 443T (numbering as usedin SEQ ID NO:1). For example, the amino acid is selected from the groupconsisting of 474V, 619P and 670G (numbering as used in SEQ ID NO:1). Inan example, the PCSK9 form is the mature form. In an example, the PCSK9form is the pro-form. In an example, the ligand also specifically bindsto form a and/or a′. In an embodiment, the ligand specifically binds toan epitope of form f PCSK9, wherein the epitope comprises at least oneamino acid that is not found in form a. In an embodiment, the ligandspecifically binds to an epitope of form c PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to an epitope of form r PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to an epitopeof form p PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to an epitope of form m PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to an epitope of form e PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to an epitope of form hPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to anepitope of form aj PCSK9, wherein the epitope comprises at least oneamino acid that is not found in form a. In an embodiment, the ligandspecifically binds to an epitope of form q PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the pro-domain of a humanPCSK9 selected from the group consisting of forms f, c, r, p, m, e, h,aj and q. In an example, the ligand also specifically binds to thepro-domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the pro-domain of form f PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform c PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the pro-domain of form r PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the pro-domain of form p PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform m PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the pro-domain of form e PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the pro-domain of form h PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform aj PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the pro-domain of form q PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the catalytic domain of ahuman PCSK9 selected from the group consisting of forms f, c, r, p, m,e, h, aj and q. In an example, the ligand also specifically binds to thecatalytic domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the catalytic domain of form f PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the catalytic domainof form c PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the catalytic domain of form r PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the catalytic domain ofform p PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the catalytic domain of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the catalytic domain of form e PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to thecatalytic domain of form h PCSK9, wherein the epitope comprises at leastone amino acid that is not found in form a. In an embodiment, the ligandspecifically binds to the catalytic domain of form aj PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the catalytic domainof form q PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a.

In an embodiment, ligand binds specifically to the C-terminal domain ofa human PCSK9 selected from the group consisting of forms f, c, r, p, m,e, h, aj and q. In an example, the ligand also specifically binds to theC-terminal domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the C-terminal domain of form f PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the C-terminal domainof form c PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the C-terminal domain of form r PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the C-terminal domain ofform p PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the C-terminal domain of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the C-terminal domain of form e PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theC-terminal domain of form h PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the C-terminal domain of form aj PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theC-terminal domain of form q PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the substrate-bindinggroove of a human PCSK9 selected from the group consisting of forms f,c, r, p, m, e, h, aj and q (see Cunningham et al., Nat Struct Mol Biol.2007 May; 14(5):413-9. Epub 2007 Apr. 15, “Structural and biophysicalstudies of PCSK9 and its mutants linked to familialhypercholesterolemia”, incorporated herein in its entirety byreference). In an example, the ligand also specifically binds to thesubstrate-binding groove of form a and/or a′. In an embodiment, theligand specifically binds to the Substrate-binding groove of form fPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form c PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form rPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form p PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form mPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form e PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form hPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form aj PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form qPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a.

Reference is made to US20120093818A1 (Amgen, Inc), the entire disclosureof which is incorporated herein. This patent application disclosesrelevant ligands for use in the present invention, as well as examplesand methods of producing and testing ligands that can be used withreference to the present invention.

In an example, the ligand is or comprises an antibody disclosed in Table2 of US20120093818A1 (Amgen, Inc) or is a PCSK9-binding derivativethereof

In an embodiment, the PCSK9-binding ligand of the invention is selectedfrom the antigen binding proteins disclosed in US20120093818A1 (Amgen,Inc), eg, in paragraphs [0009] to and [0058] to [0063] ofUS20120093818A1; all of these disclosures (including the sequences ofsuch proteins) are incorporated herein by reference as though explicitlyrecited herein and for possible inclusion in one or more claims or foruse in the present invention.

In this paragraph SEQ ID NOs are those as appearing in US20120093818A1(Amgen, Inc) and these sequences are incorporated herein by reference asthough explicitly recited herein and for possible inclusion in one ormore claims or for use in the present invention. In some aspects, theligand of the invention comprises an isolated antigen binding proteinthat binds PCSK9 comprising: A) one or more heavy chain complementarydetermining regions (CDRHs) selected from the group consisting of: (i) aCDRH1 from a CDRH1 in a sequence selected from the group consisting ofSEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii)a CDRH2 from a CDRH2 in a sequence selected from the group consisting ofSEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60;(iii) a CDRH3 from a CDRH3 in a sequence selected from the groupconsisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54,55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81,and 60; and (iv) a CDRH of (i), (ii), and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more than 4amino acids; B) one or more light chain complementary determiningregions (CDRLs) selected from the group consisting of: (i) a CDRL1 froma CDRL1 in a sequence selected from the group consisting of SEQ ID NO:5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (ii) a CDRL2 from aCDRL2 in a sequence selected from the group consisting of SEQ ID NO: 5,7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (iii) a CDRL3 from aCDRL3 in a sequence selected from the group consisting of SEQ ID NO: 5,7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and (iv) a CDRL of(i), (ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 4 amino acids; or C) one or moreheavy chain CDRHs of A) and one or more light chain CDRLs of B). In someembodiments, the isolated antigen binding protein comprises at least oneCDRH of A) and at least one CDRL of B). In some embodiments, theisolated antigen binding protein comprises at least two CDRH of A) andat least two CDRL of B). In some embodiments, the isolated antigenbinding protein comprises said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 andCDRL3. In some embodiments, the CDRH of A) is selected from at least oneof the group consisting of: (i) a CDRH1 amino acid sequence selectedfrom the CDRH1 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; (ii) a CDRH2 amino acid sequence selectedfrom the CDRH2 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; (iii) a CDRH3 amino acid sequence selectedfrom the CDRH3 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; and (iv) a CDRH of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 2 amino acids. In addition, the CDRL of B) is selectedfrom at least one of the group consisting of: (i) a CDRL1 amino acidsequence selected from the CDRL1 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; (ii) a CDRL2 amino acidsequence selected from the CDRL2 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; (iii) a CDRL3 amino acidsequence selected from the CDRL3 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; and (iv) a CDRL of (i),(ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 2 amino acids; or C) one or moreheavy chain CDRHs of A) and one or more light chain CDRLs of B. In someembodiments, the CDRH of A) is selected from at least one of the groupconsisting of: (i) a CDRH1 amino acid sequence of the CDRH1 amino acidsequence in SEQ ID NO: 67; (ii) a CDRH2 amino acid sequence of the CDRH2amino acid sequence in SEQ ID NO: 67; (iii) a CDRH3 amino acid sequenceof the CDRH3 amino acid sequence in SEQ ID NO: 67; and (iv) a CDRH of(i), (ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 2 amino acids; said CDRL of B)is selected from at least one of the group consisting of: (i) a CDRL1amino acid sequence of the CDRL1 amino acid sequence in SEQ ID NO: 12;(ii) a CDRL2 amino acid sequence of the CDRL2 amino acid sequence in SEQID NO: 12; (iii) a CDRL3 amino acid sequence of the CDRL3 amino acidsequence in SEQ ID NO: 12; and (iv) a CDRL of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 2 amino acids; or C) one or more heavy chain CDRHs of A)and one or more light chain CDRLs of B). In some embodiments, theantigen binding protein comprises A) a CDRH1 of the CDRH1 sequence inSEQ ID NO: 67, a CDRH2 of the CDRH2 sequence in SEQ ID NO: 67, and aCDRH3 of the CDRH3 sequence in SEQ ID NO: 67, and B) a CDRL1 of theCDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence in SEQ IDNO: 12, and a CDRL3 of the CDRL3 sequence in SEQ ID NO: 12. In someembodiments, the antigen binding protein comprises a heavy chainvariable region (VH) having at least 80% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 74, 85,71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62,89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or a light chainvariable region (VL) having at least 80% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9,10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32,33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, the VHhas at least 90% sequence identity with an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52,51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77,78, 83, 69, 81, and 60, and/or the VL has at least 90% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In someembodiments, the VH is selected from the group consisting of SEQ ID NO:74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91,64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL isselected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36,37, 38, 39, 40, 42, 44, and 46.

In an example of any aspect of the invention, the PCSK9-targeting orbinding ligand comprises or consists of AMG145 or 31H4, 16F12, 11F1, 8A3or 21B12 disclosed in US20120093818A1 (Amgen, Inc) or an antibodycomprising the variable domains of AMG145, 31H4, 16F12, 11F1, 8A3 or21B12, the disclosures of which (including sequences) are incorporatedherein by reference as though explicitly recited herein and for possibleinclusion in one or more claims or for use in the present invention.Preferably, the PCSK9-targeting or binding ligand comprises or consistsof AMG145.

In an example, the AMG145 or other ligand of the invention isglycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos orHek293 cell). In an example, the ligand of the invention is produced inCHO.

Reference is made to US20110065902A1 (Regeneron Pharmaceuticals, Inc),the entire disclosure of which is incorporated herein. This patentapplication discloses relevant ligands for use in the present invention,as well as examples and methods of producing and testing ligands anddetermining medical efficacy that can be used with reference to thepresent invention.

Reference is made to the following PCT applications, the entiredisclosures of which are incorporated herein. These disclose relevantligands for use in the present invention, as well as examples andmethods of producing and testing ligands and determining medicalefficacy that can be used with reference to the present invention.

WO2008057457 WO2008057458 WO2008057459 WO2008063382 WO2008133647WO2009100297 WO2009100318 WO2011037791 WO2011053759 WO2011053783WO2008125623 WO2011072263 WO2009055783 WO2010029513 WO2011111007WO2010077854

Antibody ligands to PCSK9 are described in, for example, WO 2008/057457,WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, and US2008/0008697; each of which is incorporated by reference herein in itsentirety.

In an example, the ligand is or comprises an antibody disclosed in theExamples of US20110065902A1 (eg, 316P or 300N) or is a PCSK9-bindingderivative thereof. All of these disclosures (including the sequences ofsuch proteins and corresponding nucleotide sequences) are incorporatedherein by reference as though explicitly recited herein and for possibleinclusion in one or more claims or for use in the present invention. Inan embodiment, the ligand is or comprises the variable domains ofantibody 316P or 300N disclosed in US20110065902A1 or is (or comprises)such antibody or a PCSK9-binding derivative thereof

In an embodiment, the ligand is or comprises the variable domains ofantibody alirocumab or SAR236553/REGN727 (Sanofi Aventis/Regeneron) oris (or comprises) such antibody or a PCSK9-binding derivative thereof.In an example, the alirocumab is glycosylated, eg, has humanglycosylation (eg, produced by a CHO, Cos or Hek293 cell). Preferably,the ligand is alirocumab or SAR236553/REGN727.

In an embodiment, the ligand is or comprises the variable domains ofantibody evolocumab or is (or comprises) such antibody or aPCSK9-binding derivative thereof. In an example, the antibody isglycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos orHek293 cell). Preferably, the ligand is evolocumab.

In an embodiment, the ligand is selected from evolocumab, 1D05-IgG2(Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat) and alirocumab.

In an embodiment, the ligand is selected from the following (sequencesand definitions as per US2011/0065902, incorporated herein byreference):—

1. An antibody or antigen-binding fragment thereof which specificallybinds hPCSK9, wherein the antibody or antigen-binding fragment comprisesthe heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pairhaving SEQ ID NOs: 218/226.2. The antibody or antigen-binding fragment of concept 1 comprisingheavy and light chain CDR amino acid sequences having SEQ ID NOs: 220,222, 224, 228, 230 and 232.3. The antibody or antigen-binding fragment of concept 2 comprising anHCVR having the amino acid sequence of SEQ ID NO: 218 and an LCVR havingthe amino acid sequence of SEQ ID NO: 226.4. An antibody or antigen-binding fragment thereof which binds to thesame epitope on hPCSK9 as an antibody comprising heavy and light chainCDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and232.5. An antibody or antigen-binding fragment thereof which competes forbinding to hPCSK9 with an antibody comprising heavy and light chain CDRamino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

In an embodiment, the ligand is selected from the following (sequencesand definitions as per US2012/0093818, incorporated herein byreference):—

1. An isolated neutralizing antigen binding protein that binds to aPCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1,wherein the neutralizing antigen binding protein decreases the LDLRlowering effect of PCSK9 on LDLR, wherein the antigen binding proteincomprises a light chain comprising an amino acid sequence of SEQ ID NO:46, and wherein the antigen binding protein comprises a heavy chaincomprising an amino acid sequence of SEQ ID NO: 60.2. The isolated neutralizing antigen binding protein of concept 2,wherein the antigen binding protein is a LDLR non-competitiveneutralizing antigen binding protein.3. The isolated neutralizing antigen binding protein of concept 2,wherein the antigen binding protein is a LDLR competitive neutralizingantigen binding protein.4. An antigen binding protein that selectively binds to PCSK9, whereinsaid antigen binding protein binds to PCSK9 with a Kd that is less than100 pM.5. An antigen binding protein that binds to a PCSK 9 protein of SEQ IDNO: 303 in a first manner, wherein the antigen binding protein binds toa variant of PCSK9 in a second manner, wherein said PCSK9 variant has atleast one point mutation at a position selected from the groupconsisting of: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390,413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 ofSEQ ID NO: 303, wherein the first manner comprises a first EC50, a firstBmax, or a first EC50 and a first Bmax, wherein the second mannercomprises a second EC50, a second Bmax, or a second EC50 and a secondBmax, and wherein a value for the first manner is different from a valuefor the second manner, and wherein the antigen binding protein comprisesa light chain comprising an amino acid sequence of SEQ ID NO: 46, andwherein the antigen binding protein comprises a heavy chain comprisingan amino acid sequence of SEQ ID NO: 60.6. The antigen binding protein of concept 6, wherein the first mannercomprises a first Bmax, wherein the second manner comprises a secondBmax that is different from the first Bmax, and wherein said PCSK9variant has at least one point mutation selected from the groupconsisting of: D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R,R519E, H521R, and Q554R.7. The antigen binding protein of concept 6, wherein the antigen bindingprotein binds to PCSK9 at a location that overlaps with a location thatLDLR binds to PCSK9.8. A method of making an antigen binding protein that binds to a PCSK9protein comprising the amino acid sequence of SEQ ID NO: 1, wherein theantigen binding protein decreases the LDLR lowering effect of PCSK9 onLDLR, said method comprising: providing a host cell comprising a nucleicacid sequence that encodes the antigen binding protein; and maintainingthe host cell under conditions in which the antigen binding protein isexpressed, wherein the antigen binding protein comprises a light chaincomprising an amino acid sequence of SEQ ID NO: 46, and wherein theantigen binding protein comprises a heavy chain comprising an amino acidsequence of SEQ ID NO: 60.9. A method for treating or preventing a condition associated withelevated serum cholesterol levels in a subject, said method comprisingadministering to a subject in need thereof an effective amount of anisolated neutralizing antigen binding protein simultaneously orsequentially with an agent that elevates the availability of LDLRprotein, wherein the isolated antigen binding protein binds to a PCSK9protein comprising the amino acid sequence of SEQ ID NO: 1, wherein theneutralizing antigen binding protein decreases the LDLR lowering effectof PCSK9 on LDLR, wherein the antigen binding protein comprises a lightchain comprising an amino acid sequence of SEQ ID NO: 46, and whereinthe antigen binding protein comprises a heavy chain comprising an aminoacid sequence of SEQ ID NO: 60.10. The method of concept 10, wherein the agent that elevates theavailability of LDLR protein comprises a statin.11. An antigen binding protein that binds to PCSK9, wherein when theantigen binding protein is bound to PCSK9, the antibody is positioned 8angstroms or less from at least one of the following residues of PCSK9:S153, S188, 1189, Q190, S191, D192, R194, E197, G198, R199, V200, D224,R237, D238, K243, S373, D374, S376, T377, F379, 1154, T187, H193, E195,1196, M201, V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,C375, or C378, wherein the antigen binding protein comprises a lightchain comprising an amino acid sequence of SEQ ID NO: 46, and whereinthe antigen binding protein comprises a heavy chain comprising an aminoacid sequence of SEQ ID NO: 60.

The ligand can be used for the treatment, therapy, prophylaxis and/ordiagnosis of one or more diseases or conditions or susceptibilitythereto, wherein such diseases or conditions comprise those disclosed inUS20120093818A1 (Amgen, Inc) and US20110065902A1 (RegeneronPharmaceuticals, Inc), eg, a disease or condition disclosed inparagraphs [0375] to [0383] of US20120093818A1, which disclosure isincorporated herein by reference in its entirety for inclusion in onemore claims herein.

The ligand can be administered to a human characterised as described inUS20120093818A1 (Amgen, Inc) or US20110065902A1.

The ligand can be administered in a form or combination disclosed inUS20120093818A1 (Amgen, Inc) or US20110065902A1, which disclosure isincorporated herein by reference. For example, the ligand with a drug,excipient, diluent or carrier as described in US20120093818A1 (Amgen,Inc) or US20110065902A1 (eg, as disclose in paragraphs [0384] to [0412]of US20120093818A1), which disclosure is incorporated herein byreference, and the present invention also relates to the correspondingpharmaceutical compositions comprising the combination of a ligand ofthe invention and such a further agent.

The ligand can be used in a method of diagnosis as set out inUS20120093818A1 (Amgen, Inc) or US20110065902A1, eg, in paragraphs[0413] to [0415] of US20120093818A1 which disclosure is incorporatedherein by reference.

Diagnostic Applications

In some embodiments, the ligand of the invention is a diagnostic tool.The ligand can be used to assay the amount of PCSK9 present in a sampleand/or subject. As will be appreciated by one of skill in the art, suchligands need not be neutralizing ligands. In some embodiments, thediagnostic ligand is not a neutralizing ligand. In some embodiments, thediagnostic ligand binds to a different epitope than a neutralizingligand binds to. In some embodiments, the two ligands do not competewith one another.

In some embodiments, the ligands of the invention are used or providedin an assay kit and/or method for the detection of PCSK9 in mammaliantissues or cells in order to screen/diagnose for a disease or disorderassociated with changes in levels of PCSK9. The kit comprises a ligandthat binds PCSK9 and means for indicating the binding of the ligand withPCSK9, if present, and optionally PCSK9 protein levels. Various meansfor indicating the presence of a ligand can be used. For example,fluorophores, other molecular probes, or enzymes can be linked to theligand and the presence of the ligand can be observed in a variety ofways. The method for screening for such disorders can involve the use ofthe kit, or simply the use of one of the disclosed ligands and thedetermination of whether the ligand binds to PCSK9 in a sample. As willbe appreciated by one of skill in the art, high or elevated levels ofPCSK9 will result in larger amounts of the ligand binding to PCSK9 inthe sample. Thus, degree of ligand binding can be used to determine howmuch PCSK9 is in a sample. Subjects or samples with an amount of PCSK9that is greater than a predetermined amount (e.g., an amount or rangethat a person without a PCSK9 related disorder would have) can becharacterized as having a PCSK9 mediated disorder. In some embodiments,the invention provides a method wherein the ligand is administered to asubject taking a statin, in order to determine if the statin hasincreased the amount of PCSK9 in the subject.

In some embodiments, the ligand is a non-neutralizing ligand and is usedto determine the amount of PCSK9 in a subject receiving an ABP and/orstatin treatment.

In some embodiments, the ligand of the invention can specifically bindhuman PCSK9 (eg, one, two or more rare variant forms disclosed herein)and is characterized by at least one of: (i) capable of reducing serumtotal cholesterol at least about 25-35% and sustaining the reductionover at least a 24 day period relative to a predose level; (ii) capableof reducing serum LDL cholesterol at least about 65-80% and sustainingthe reduction over at least a 24 day period relative to a predose level;(iii) capable of reducing serum LDL cholesterol at least about 40-70%and sustaining the reduction over at least a 60 or 90 day periodrelative to a predose level; (iv) capable of reducing serum triglycerideat least about 25-40% relative to predose level; (v) does not reduceserum HDL cholesterol or reduces serum HDL cholesterol no more than 5%relative to predose level. In some embodiments, an isolated nucleic acidmolecule is provided and it encodes the ligand. In some embodiments anexpression vector is provided and comprises the nucleic acid molecule.In some embodiments, a pharmaceutical composition is provided and it cancomprise the ligand and a pharmaceutically acceptable carrier. In someembodiments, a method is provided for treating a disease or conditionwhich is ameliorated, improved, inhibited or prevented with a PCSK9antagonist ligand of the invention. The method can compriseadministering a therapeutic amount of the pharmaceutical composition orligand to a subject in need thereof. In some embodiments, the subject isa human subject suffering from hypercholesterolemia, hyperlipidemia,indicated for LDL apheresis, identified as heterozygous for FamilialHypercholesterolemia, statin intolerant. statin uncontrolled, at riskfor developing hypercholesterolemia, dyslipidemia, cholestatic liverdisease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosisand cardiovascular diseases. In some embodiments, a method of providinga treatment or therapy is provided to a subject. In some embodiments,the method comprises reducing serum cholesterol at least about 40-70%over at least 60 to 90 days. In some embodiments, a method of receivingtreatment or therapy is provided, the method can comprise receiving aligand thereof at a frequency of once every 60 to 90 days.

In one aspect, the invention provides a ligand of the invention which isor comprises an human antibody or antigen-binding fragment of a humanantibody that specifically binds and inhibits human proproteinconvertase subtilisin/kexin type 9 (hPCSK9, eg, one, two or more rarevariant forms disclosed herein and optionally form a and/or form a′),characterized by the ability to reduce serum LDL cholesterol in a humanby 40-80% over a 24, 60 or 90 day period relative to predose levels,with little or no reduction in serum HDL cholesterol and/or with littleor no measurable effect on liver function, as determined by ALT and ASTmeasurements.

In one embodiment, the ligand of the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum total cholesterol at least about 25-35%and sustaining the reduction over at least a 24 day period relative to apredose level, preferably the reduction in serum total cholesterol is atleast about 30-40%;(ii) capable of reducing serum LDL cholesterol at least about 65-80% andsustaining the reduction over at least a 24 day period relative to apredose level;(iii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;(iv) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose level.

See US2011/0065902 for definitions of these terms and optional features,the disclosure of which is incorporated herein by reference in itsentirety.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum LDL cholesterol at least about 40-70% andsustaining the reduction over at least a 60 or 90 day period relative toa predose level;(ii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;(iii) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose level.

In one embodiment, the antibody or antigen-binding fragment ischaracterized as exhibiting an enhanced binding affinity (KD) for hPCSK9at pH 5.5 relative to the KD at pH 7.4, as measured by plasmon surfaceresonance. In a specific embodiment, the antibody or fragment thereofexhibits at least a 20-fold, at least a 40-fold or at least a 50-foldenhanced affinity for PCSK9 at an acidic pH relative to a neutral pH, asmeasured by surface plasmon resonance

In one embodiment, the antibody or antigen-binding fragment ischaracterized as not exhibiting an enhanced binding affinity for PCSK9at an acidic pH relative to a neutral pH, as measured by surface plasmonresonance. In a specific embodiment, the antibody or fragment thereofexhibits a decreased binding affinity at an acidic pH.

In another embodiment, the antibody or antigen-binding fragment bindshuman, human GOF mutation D374Y, cynomolgus monkey, rhesus monkey,mouse, rat and hamster PCSK9.

In one embodiment, the antibody or antigen-binding fragment binds humanand monkey PCSK9, but does not bind mouse, rat or hamster PCSK9.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody comprising one or more of aheavy chain variable region (HCVR), light chain variable region (LCVR),HCDR1, HCDR2, HCDR3 disclosed in any of paragraphs [023]-[037] ofUS2011/0065902, the disclosures of which are incorporated herein byreference.

In a related embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody which specifically binds hPCSK9,wherein the antibody or fragment comprises heavy and light chain CDRdomains contained within heavy and light chain sequence pairs selectedfrom the group consisting of SEQ ID NO (using the sequence numbering inUS2011/0065902): 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68,70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140,142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202,210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264,266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332,334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394,402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456,458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524,526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586,594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648,650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716,718/720, 722/730, 738/740 and 742/744. In one embodiment, the CDRsequences are contained within HCVR and LCVR selected from the aminoacid sequence pairs of SEQ ID NO: 50/58, 66/68, 70/72, 74/82, 90/92,94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 238/240, 242/250,258/260, 262/264, 314/322, 330/332 and 334/336. In more specificembodiments, the CDR sequences are comprised within HCVR/LCVR sequencesselected from SEQ ID NO: 90/92 or 218/226.

In an example, the invention features a pharmaceutical compositioncomprising a ligand of the invention, wherein the ligand comprises orconsists of a recombinant human antibody or fragment thereof whichspecifically binds hPCSK9 and a pharmaceutically acceptable carrier. Inone embodiment, the invention features a composition which is acombination of a ligand of the invention (eg, an antibody orantigen-binding fragment of an antibody), and a second therapeuticagent. The second therapeutic agent may be any agent that isadvantageously combined with the ligand of the invention, for example,an agent capable of inducing a cellular depletion of cholesterolsynthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A(CoA) reductase, such as, for example, cerovastatin, atorvastatin,simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin,pravastatin, etc; capable of inhibiting cholesterol uptake and or bileacid re-absorption; capable of increasing lipoprotein catabolism (suchas niacin); and/or activators of the LXR transcription factor that playsa role in cholesterol elimination such as 22-hydroxycholesterol.

In an example, the invention provides a method for inhibiting hPCSK9activity using the anti-PCSK9 ligand of the invention (eg, an antibodyor antigen-binding portion of the antibody of the invention), whereinthe therapeutic methods comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an antibodyor antigen-binding fragment of an antibody of the invention. Thedisorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by removal, inhibition or reductionof PCSK9 activity. Specific populations treatable by the therapeuticmethods of the invention include subjects indicated for LDL apheresis,subjects with PCSK9-activating mutations (gain of function mutations,“GOF”), subjects with heterozygous Familial Hypercholesterolemia (heFH);subjects with primary hypercholesterolemia who are statin intolerant orstatin uncontrolled; and subjects at risk for developinghypercholesterolemia who may be preventably treated. Other indicationsinclude dyslipidemia associated with secondary causes such as Type 2diabetes mellitus, cholestatic liver diseases (primary biliarycirrhosis), nephrotic syndrome, hypothyroidism, obesity; and theprevention and treatment of atherosclerosis and cardiovascular diseases.

In specific embodiments of the method of the invention, the ligand ofthe invention (eg, anti-hPCSK9 antibody or antibody fragment of theinvention) is useful to reduce elevated total cholesterol, non-HDLcholesterol, LDL cholesterol, and/or apolipoprotein B (apolipoproteinB100).

The ligand (eg, antibody or antigen-binding fragment) of the inventionmay be used alone or in combination with a second agent, for example, anHMG-CoA reductase inhibitor and/or another lipid lowering drug.

Treatment Population

The invention provides therapeutic methods for treating a human patientin need of a composition or ligand of the invention. While modificationsin lifestyle and conventional drug treatment are often successful inreducing cholesterol levels, not all patients are able to achieve therecommended target cholesterol levels with such approaches. Variousconditions, such as familial hypercholesterolemia (FH), appear to beresistant to lowering of LDL-C levels in spite of aggressive use ofconventional therapy. Homozygous and heterozygous familialhypercholesterolemia (hoFH, heFH) is a condition associated withpremature atherosclerotic vascular disease. However, patients diagnosedwith hoFH are largely unresponsive to conventional drug therapy and havelimited treatment options. Specifically, treatment with statins, whichreduce LDL-C by inhibiting cholesterol synthesis and upregulating thehepatic LDL receptor, may have little effect in patients whose LDLreceptors are non-existent or defective. A mean LDL-C reduction of onlyless than about 20% has been recently reported in patients withgenotype-confirmed hoFH treated with the maximal dose of statins. Theaddition of ezetimibe 10 mg/day to this regimen resulted in a totalreduction of LDL-C levels of 27%, which is still far from optimal.Likewise, many patients are statin non-responsive, poorly controlledwith statin therapy, or cannot tolerate statin therapy; in general,these patients are unable to achieve cholesterol control withalternative treatments. There is a large unmet medical need for newtreatments that can address the short-comings of current treatmentoptions.

Specific populations treatable by the therapeutic methods of theinvention include patients indicated for LDL apheresis, subjects withPCSK9-activating (GOF) mutations, heterozygous FamilialHypercholesterolemia (heFH); subjects with primary hypercholesterolemiawho are statin intolerant or statin uncontrolled; and subjects at riskfor developing hypercholesterolemia who may be preventably treated.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-PCSK9 ligands, antibodies or antigen-binding fragments thereof ofthe present invention. The administration of therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the ligand, eg, antibody, of the present invention isused for treating various conditions and diseases associated with PCSK9,including hypercholesterolemia, disorders associated with LDL andapolipoprotein B, and lipid metabolism disorders, and the like, in anadult patient, it is advantageous to intravenously administer the ligandor antibody of the present invention normally at a single dose of about0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, thus the compositioninvention provides the ligand by e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al.(1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecomposition may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPENT™, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

The invention provides therapeutic methods in which the ligand, eg,antibody or antibody fragment, of the invention is useful to treathypercholesterolemia associated with a variety of conditions involvinghPCSK9. The anti-PCSK9 ligands, eg, antibodies or antibody fragments, ofthe invention are particularly useful for the treatment ofhypercholesterolemia and the like. Combination therapies may include theanti-PCSK9 ligand of the invention with, for example, one or more of anyagent that (1) induces a cellular depletion of cholesterol synthesis byinhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase,such as cerivastatin, atorvastatin, simvastatin, pitavastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin; (2) inhibitscholesterol uptake and or bile acid re-absorption; (3) increaselipoprotein catabolism (such as niacin); and activators of the LXRtranscription factor that plays a role in cholesterol elimination suchas 22-hydroxycholesterol or fixed combinations such as ezetimibe plussimvastatin; a statin with a bile resin (e.g., cholestyramine,colestipol, colesevelam), a fixed combination of niacin plus a statin(e.g., niacin with lovastatin); or with other lipid lowering agents suchas omega-3-fatty acid ethyl esters (for example, omacor).

Tailoring Antibodies to Rare PCSK9 Variant Profile

As outline above, the invention includes the possibility to tailortreatment of humans further by selecting antibody-based ligands withvariable domains based on gene segments commonly found in humans of theethnic populations where the variant PCSK9 forms are found to meet theselection criteria of the invention. An example is provided below forligands comprising antibody VH domains derived from recombination ofhuman VH3-23.

The inventor analysed the frequencies and distribution of various humanVH3-23 alleles and realised the desirability of using ligands based onhuman VH3-23 alleles comprising SNP rs56069819. This SNP corresponds toa change from leucine at position 24 in the encoded protein sequence toa valine at that position (L24V change) and the SNP is at coordinate106725482 on human chromosome 14.

FIG. 2 shows the cumulative allele frequency distribution across the1000 Genomes Project database of human VH3-23 alleles comprising SNPrs56069819 (such alleles denonted “C” and the most frequent allele(which does not comprise this SNP) denoted “A”). The figure shows thatVH3-23 alleles comprising SNP rs56069819 are present at a cumulativefrequency of 11% across all human ethnic populations taken as a whole,whereas in certain specific human ethnic sub-populations (ASW, LWK, YRI,CEU and GBR) such alleles are present at an above-average cumulativefrequency. Indicated in the figure are those human PCSK9 variant forms(marked “Variants”) that are found in the various sub-populations withabove-average occurrence of human VH3-23 alleles comprising SNPrs56069819. Table 7 shows the VH3-23 variants and the SNPs that theycomprise, as well as their cumulative allele frequencies as found in the1000 Genomes Project database.

Notably, human VH3-23 alleles comprising SNP rs56069819 were found inthe CEU population at a frequency that is almost double the frequency of11% for all populations. For the ASW and YRI populations the frequencywas over a quarter of the population. Thus, the invention advantageouslyenables one to select a ligand comprising an antibody or antibodyfragment, wherein the antibody or fragment comprises a VH domain derivedfrom the recombination of a human VH gene segment, a human D genesegment and a human JH gene segment, the VH gene segment comprising anucleotide sequence that comprises SNP rs56069819 (dbSNP numbering,build number as recited above).

In an example, one can tailor the treatment further by selecting such aligand that specifically binds to a human PCSK9 selected from forms: f,c, m, e, h, p, q and aj, such forms being those appearing in humanpopulations ASW, LWK, YRI, CEU and GBR.

In an example, the VH gene segment is VH3-23*04, which is a commonlyfound variant that comprises SNP rs56069819 in human populations ASW,LWK, YRI, CEU and GBR.

In an example, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human that expresses a humanPCSK9 selected from forms: f, c, m, e, h, p, q and aj.

In an example, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of ASW, LWK, YRI, CEU orGBR ancestry.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of ASW ancestry, whereinthe human expresses a PCSK9 selected from f, c, m, e, h, p and q or thehuman comprises a corresponding nucleotide or amino acid sequence as setout in Table 6. Optionally this ligand comprises a VH domain derivedfrom recombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of LWK ancestry, whereinthe human expresses a PCSK9 selected from f, c, m, e and h or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of YRI ancestry, whereinthe human expresses a PCSK9 selected from f, c, m, e and h or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of CEU ancestry, whereinthe human expresses a PCSK9 selected from f, c, p and aj or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of GBR ancestry, whereinthe human expresses a PCSK9 selected from f, c and p or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an example, the ligand is alirocumab.

In other embodiments, as explained more fully above, the inventionprovides for ligands which are tailored to the human recipient'sgenotype and/or phenotype based on alternative human VH gene segments,or on Vκ, Vλ or constant region gene segments (see further Table 9 forrepresentative variants).

For example, the ligand of the invention comprises or consists of anantibody that comprises a VH domain that is derived from therecombination of a human VH gene segment, a human D gene segment and ahuman JH gene segment, wherein the VH gene segment is selected from thegroup consisting of (i) IGHV1-18*01 and the genome of the humancomprises a human IGHV1-18*01 nucleotide sequence or the human expressesantibodies comprising variable domains derived from the recombination ofhuman IGHV1-18*01; or (ii) IGVH1-46*01 and the genome of the humancomprises a human IGHV1-46*01 nucleotide sequence or the human expressesantibodies comprising variable domains derived from the recombination ofhuman IGHV1-46*01.

For example, the ligand of the invention comprises or consists of anantibody that comprises a VL domain that is derived from therecombination of a human VL gene segment and a human JL gene segment,wherein the VL gene segment is selected from the group consisting of (i)IGKV4-1*01 and the genome of the human comprises a human IGKV4-1*01nucleotide sequence or the human expresses antibodies comprisingvariable domains derived from the recombination of human IGKV4-1*01;(ii) IGLV2-14*01 and the genome of the human comprises a humanIGLV2-14*01 nucleotide sequence or the human expresses antibodiescomprising variable domains derived from the recombination of humanIGLV2-14*01; or (iii) IGKV1-13*02 and the genome of the human comprisesa human IGKV1-13*02 nucleotide sequence or the human expressesantibodies comprising variable domains derived from the recombination ofhuman IGKV1-13*02.

For example, the inventor identified the possibility of addressing therarer IGH-gamma-1 SNPs 204D (observed cumulative frequency of 0.296) and206L (observed cumulative frequency of 0.283) individually or incombination. These residues are part of the CH3 domain, and as such theyform part of antibody Fc regions. Thus, matching of these CH3 variationswith the patient is especially beneficial for reasons as discussedabove. Thus, in this example the ligand of the invention comprises orconsists of an antibody that comprises a human gamma-1 heavy chainconstant region that comprises an Asp corresponding to position 204 ofSEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42and wherein the genome of the human comprises a gamma-1 heavy chainconstant region nucleotide sequence that encodes such an Asp or Leu orthe human expresses antibodies comprising human gamma-1 constant regionscomprising such an Asp or Leu. An example of such a ligand isalirocumab.

In another example, the inventor identified the possibility ofaddressing IGH-gamma-2 SNPs. This included consideration of Fc regionvariation—in this respect, the inventor focused on positions 161 and 257which are in the Fc region. Thus, in this example the ligand of theinvention comprises or consists of an antibody that comprises a humangamma-2 heavy chain constant region that comprises an amino acidselected from the group consisting of a Pro corresponding to position 72of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44,a Phe corresponding to position 76 of SEQ ID NO: 44, a Val correspondingto position 161 of SEQ ID NO: 44 and an Ala corresponding to position257 of SEQ ID NO: 44; and wherein the genome of the human comprises agamma-2 heavy chain constant region nucleotide sequence that encodessuch a selected amino acid or the human expresses antibodies comprisinghuman gamma-2 constant regions comprising such a selected amino acid. Anexample of such a ligand is evolocumab or bococizumab.

In another example, the inventor addressed human kappa constant regionvariation. Thus, in this example the ligand of the invention comprisesor consists of an antibody that comprises a human kappa light chainconstant region that comprises a Val corresponding to position 84 of SEQID NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50; andwherein the genome of the human comprises a kappa light chain constantregion nucleotide sequence that encodes such a Val or Cys or the humanexpresses antibodies comprising human kappa light chain constant regionscomprising such a Val or Cys. An example of such a ligand is alirocumabor bococizumab.

In another example, the inventor addressed human lambda constant regionvariation. Thus, in this example the ligand of the invention comprisesor consists of an antibody that comprises a human IGLC2*01 light chainconstant region; and wherein the genome of the human comprises a humanIGLC2*01 nucleotide sequence or the human expresses antibodiescomprising human light chain IGLC2*01 constant regions. An example ofsuch a ligand is evolocumab.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   -   1. An antibody or antibody fragment for use in a method of        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof, wherein the        antibody or fragment comprises a human gamma heavy chain        constant region that comprises a first amino acid that is        encoded by a human gamma heavy chain constant region gene        segment SNP, and the antibody or fragment specifically binds a        proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid        sequence that comprises a C-terminal domain comprising a        mutation I474V or E670G in SEQ ID NO:1, wherein the human        comprises a nucleotide sequence encoding said PCSK9 amino acid        sequence and comprises a human gamma heavy chain constant region        gene segment comprising said SNP, or the human expresses        antibodies comprising human gamma constant regions comprising        said first amino acid.        -   In an alternative, paragraph 1 provides:—        -   An antibody or antibody fragment for use in a method of            reducing cholesterol level or maintaining previously reduced            cholesterol level in a human in need thereof, wherein the            antibody or fragment comprises a human gamma-1 heavy chain            constant region that comprises an Asp corresponding to            position 204 of SEQ ID NO: 42 or a Leu corresponding to            position 206 of SEQ ID NO: 42, and the antibody or fragment            specifically binds a proprotein convertase subtilisin/kexin            type 9 (PCSK9) amino acid sequence that comprises a            C-terminal domain comprising a mutation I474V or E670G in            SEQ ID NO:1, wherein the human comprises a nucleotide            sequence encoding said amino acid sequence and comprises an            IGHG1*01 human heavy chain constant region gene segment, or            the human expresses antibodies comprising human gamma-1            constant regions comprising such an Asp and Leu.        -   In an embodiment, said mutation is I474V. In another            embodiment, said mutation is E670G.        -   An example provides:—        -   An antibody or antibody fragment for use in a method of            reducing cholesterol level or maintaining previously reduced            cholesterol level in a human in need thereof, wherein the            antibody or fragment comprises a human gamma-1 heavy chain            constant region that comprises an Asp corresponding to            position 204 of SEQ ID NO: 42 and a Leu corresponding to            position 206 of SEQ ID NO: 42, and the antibody or fragment            specifically binds a proprotein convertase subtilisin/kexin            type 9 (PCSK9) amino acid sequence that comprises a            C-terminal domain comprising a mutation I474V in SEQ ID            NO:1, wherein the human comprises a nucleotide sequence            encoding said amino acid sequence and comprises an IGHG1*01            human heavy chain constant region gene segment. Optionally,            the antibody or fragment comprises an IGHG1*01 human heavy            chain constant region.        -   Another example provides:—        -   An antibody or antibody fragment for use in a method of            reducing cholesterol level or maintaining previously reduced            cholesterol level in a human in need thereof, wherein the            antibody or fragment comprises a human gamma-1 heavy chain            constant region that comprises an Asp corresponding to            position 204 of SEQ ID NO: 42 and a Leu corresponding to            position 206 of SEQ ID NO: 42, and the antibody or fragment            specifically binds a proprotein convertase subtilisin/kexin            type 9 (PCSK9) amino acid sequence that comprises a            C-terminal domain comprising a mutation E670G in SEQ ID            NO:1, wherein the human comprises a nucleotide sequence            encoding said amino acid sequence and comprises an IGHG1*01            human heavy chain constant region gene segment. Optionally,            the antibody or fragment comprises an IGHG1*01 human heavy            chain constant region.        -   In an example, said antibody or antibody fragment has been            determined to specifically bind a proprotein convertase            subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal            domain comprising a mutation I474 or E670G in SEQ ID NO: 1,            wherein the antibody or fragment comprises a human gamma-1            heavy chain constant region that comprises an Asp            corresponding to position 204 of SEQ ID NO: 42 and a Leu            corresponding to position 206 of SEQ ID NO: 42 and wherein            said human comprises (i) an IGHG1*01 human heavy chain            constant region gene segment and (ii) a nucleotide sequence            encoding said proprotein convertase subtilisin/kexin type 9            (PCSK9) that comprises a C-terminal domain comprising said            mutation I474V or E670G in SEQ ID NO: 1.    -   2. The antibody or antibody fragment of paragraph 1, wherein the        antibody comprises a human gamma-1 heavy chain constant region        that comprises an Asp corresponding to position 204 of SEQ ID        NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42.    -   3. The antibody or antibody fragment of paragraph 1 or 2,        wherein the antibody comprises an IGHG1*01 human heavy chain        constant region.    -   4. The antibody or antibody fragment of any one of paragraphs 1        to 3, wherein the human has been determined to comprise the        nucleotide sequence that encodes a PCSK9 comprising a C-terminal        domain comprising said mutation I474V or E670G in SEQ ID NO: 1        and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)        variant protein encoded by the nucleotide sequence of SEQ ID NO:        29 or 30.    -   5. The antibody or antibody fragment of any one of paragraphs 1        to 4, the method comprising the step of determining that the        human comprises the nucleotide sequence that encodes a PCSK9        comprising a C-terminal domain comprising said mutation I474V or        E670G and/or a proprotein convertase subtilisin/kexin type 9        (PCSK9) variant protein comprising said mutation I474V or E670G,        optionally, wherein the determining step is performed before        administration of the antibody to the human.    -   6. The antibody or antibody fragment of paragraph 5, wherein the        step of determining comprises assaying a biological sample from        the human for a nucleotide sequence encoding the PCSK9 that        comprises the C-terminal domain comprising the mutation I474V or        E670G in SEQ ID NO: 1.    -   7. The antibody or antibody fragment of paragraph 6, wherein the        assaying comprises contacting the biological sample with        -   a. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides that can specifically            hybridize to and identify in the biological sample a            nucleotide sequence encoding the PCSK9 that comprises the            C-terminal domain comprising the mutation I474V or E670G in            SEQ ID NO: 1 or that specifically hybridizes to an antisense            of said sequence, wherein said nucleic acid hybridizes to at            least one nucleotide present in said selected sequence which            is not present in SEQ ID NO: 28 or hybridizes to an            antisense sequence thereby forming a complex when at least            one nucleotide sequence encoding the PCSK9 that comprises            the C-terminal domain comprising the mutation I474V or E670G            in SEQ ID NO: 1 is present; and/or        -   b. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides of a nucleotide            sequence encoding the PCSK9 that comprises the C-terminal            domain comprising the mutation I474V or E670G in SEQ ID NO:            1 or comprising an antisense sequence of said contiguous            nucleotides, wherein said sequence of contiguous nucleotides            comprises at least one nucleotide present in said selected            sequence which is not present in SEQ ID NO: 28 thereby            forming a complex when the nucleotide sequence encoding the            PCSK9 that comprises a C-terminal domain comprising the            mutation I474V or E670G in SEQ ID NO: 1 is present; and            detecting the presence or absence of the complex, wherein            detecting the presence of the complex determines that the            human comprises the PCSK9 that comprises the C-terminal            domain comprising the mutation I474V or E670G in SEQ ID NO:            1.    -   8. The antibody or antibody fragment of paragraph 6 or 7,        wherein the assaying comprises nucleic acid amplification and        optionally one or more methods selected from sequencing, next        generation sequencing, nucleic acid hybridization, and        allele-specific amplification and/or wherein the assaying is        performed in a multiplex format.    -   9. The antibody or antibody fragment of any one of paragraphs 1        to 8, wherein said antibody or antibody fragment is for        administration to a human that is or has been further determined        to be substantially resistant to statin treatment.    -   10. The antibody or antibody fragment of any one of paragraphs 1        to 9, wherein antibody or antibody fragment is for        administration to a human that is receiving or has received        statin treatment or has reduced responsiveness to statin        treatment.    -   11. The antibody or antibody fragment of paragraph 9 or 10,        wherein said antibody or antibody fragment is for administration        to the human separately or simultaneously with said statin        treatment.    -   12. The antibody or antibody fragment of any one of paragraphs 6        to 10, wherein said biological sample comprises serum, blood,        faeces, tissue, a cell, urine and/or saliva of said human.    -   13. The antibody or antibody fragment of any one of paragraphs 1        to 12, wherein said human is indicated as heterozygous for a        nucleotide sequence encoding the PCSK9 C-terminal domain        comprising a mutation I474V or E670G, optionally, wherein said        human is further indicated as comprising the nucleotide sequence        of SEQ ID NO: 28, or said human is indicated as homozygous for a        nucleotide sequence encoding the PCSK9 C-terminal domain        comprising a mutation I474V or E670G in SEQ ID NO: 1.    -   14. The antibody or antibody fragment of any one of paragraphs 1        to 13, wherein said human has been diagnosed with at least one        condition selected from a lipid disorder, hyperlipoproteinemia,        hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart        attack, a stroke, coronary heart disease, atherosclerosis,        peripheral vascular disease, claudication and high blood        pressure.    -   15. The antibody or antibody fragment of any one of paragraphs 1        to 14, wherein said antibody or antibody fragment treats or        reduces the risk in said human of at least one condition        selected from a lipid disorder, hyperlipoproteinemia,        hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart        attack, a stroke, coronary heart disease, atherosclerosis,        peripheral vascular disease, claudication and high blood        pressure.    -   16. The antibody or antibody fragment of any one of paragraphs 1        to 15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.    -   17. The antibody or antibody fragment of any one of paragraphs 1        to 16, wherein said antibody or antibody fragment is for        administration by intravenous or subcutaneous administration        and/or is comprised in an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of reducing cholesterol level or maintaining previously    reduced cholesterol level in a human in need thereof, the method    comprising administering to said human an antibody or antibody    fragment that specifically binds a proprotein convertase    subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain    comprising a mutation I474 or E670G in SEQ ID NO: 1, wherein the    antibody or fragment comprises a human gamma-1 heavy chain constant    region that comprises an Asp corresponding to position 204 of SEQ ID    NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42 and    wherein said human comprises (i) an IGHG1*01 human heavy chain    constant region gene segment, or the human expresses antibodies    comprising human gamma-1 heavy chain constant regions comprising    such an Asp and Leu and (ii) a nucleotide sequence encoding said    proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises    a C-terminal domain comprising said mutation I474V or E670G in SEQ    ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human gamma-1 heavy chain        constant region that comprises an Asp corresponding to position        204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of        SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01        human heavy chain constant region gene segment and (ii) a        nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation I474V in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises a IGHG1*01 human        heavy chain constant region.    -   Another example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation E670G in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human gamma-1 heavy chain        constant region that comprises an Asp corresponding to position        204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of        SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01        human heavy chain constant region gene segment and (ii) a        nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation E670G in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGHG1*01 human        heavy chain constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 or E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human gamma-1 heavy        chain constant region that comprises an Asp corresponding to        position 204 of SEQ ID NO: 42 and a Leu corresponding to        position 206 of SEQ ID NO: 42 and wherein said human        comprises (i) an IGHG1*01 human heavy chain constant region gene        segment and (ii) a nucleotide sequence encoding said proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising said mutation I474V or E670G in SEQ        ID NO: 1.-   2. The method of paragraph 1, comprising, before said administering,    selecting a human comprising said nucleotide sequence of (ii),    wherein the human is the human of paragraph 1.-   3. The method of paragraph 1 or 2, wherein the antibody comprises a    human gamma-1 heavy chain constant region that comprises an Asp    corresponding to position 204 of SEQ ID NO: 42 and a Leu    corresponding to position 206 of SEQ ID NO: 42.-   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises    an IGHG1*01 human heavy chain constant region.-   5. The method of any one of paragraphs 1 to 4, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation I474V    or E670G in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   6. The method of any one of paragraphs 1 to 5, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation I474V or E670G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   7. The method of paragraph 6, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation I474V or E670G in SEQ ID NO: 1.-   8. The method of paragraph 7, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   9. The method of paragraph 7 or 8, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   10. The method of any one of paragraphs 1 to 9, wherein said human    is or has been further determined to be substantially resistant to    statin treatment.-   11. The method of any one of paragraphs 1 to 10, wherein said human    is receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   12. The method of paragraph 10 or 11, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   13. The method of any one of paragraphs 7 to 9, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   14. The method of any one of paragraphs 1 to 13, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 28, or said human is indicated    as homozygous for a nucleotide sequence encoding the PCSK9    C-terminal domain comprising said mutation I474V or E670G in SEQ ID    NO: 1.-   15. The method of any one of paragraphs 1 to 14, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   17. The method of any one of paragraphs 1 to 16, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   18. The method of any one of paragraphs 1 to 17, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   1. An antibody or antibody fragment for use in a method of reducing    cholesterol level or maintaining previously reduced cholesterol    level in a human in need thereof, wherein the antibody comprises a    human gamma-2 heavy chain constant region that comprises an amino    acid selected from the group consisting of a Pro corresponding to    position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of    SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44,    a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala    corresponding to position 257 of SEQ ID NO: 44, and the antibody or    fragment specifically binds a proprotein convertase subtilisin/kexin    type 9 (PCSK9) amino acid sequence that comprises a C-terminal    domain comprising a mutation I474V or E670G in SEQ ID NO:1, wherein    the human comprises a nucleotide sequence encoding said amino acid    sequence and comprises an IGHG2*01 human heavy chain constant region    gene segment, or the human expresses antibodies comprising human    gamma-2 constant regions comprising such a Pro, Asn, Phe, Val and    Ala.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   An antibody or antibody fragment for use in a method of reducing        cholesterol level or maintaining previously reduced cholesterol        level in a human in need thereof, wherein the antibody comprises        a human gamma-2 heavy chain constant region that comprises a Pro        corresponding to position 72 of SEQ ID NO: 44, an Asn        corresponding to position 75 of SEQ ID NO: 44, a Phe        corresponding to position 76 of SEQ ID NO: 44, a Val        corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44, and the antibody        or fragment specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) amino acid sequence that        comprises a C-terminal domain comprising a mutation I474V in SEQ        ID NO:1, wherein the human comprises a nucleotide sequence        encoding said amino acid sequence and comprises an IGHG2*01        human heavy chain constant region gene segment. Optionally, the        antibody or fragment comprises an IGHG2*01 human heavy chain        constant region.    -   Another example provides:—    -   An antibody or antibody fragment for use in a method of reducing        cholesterol level or maintaining previously reduced cholesterol        level in a human in need thereof, wherein the antibody comprises        a human gamma-2 heavy chain constant region that comprises a Pro        corresponding to position 72 of SEQ ID NO: 44, an Asn        corresponding to position 75 of SEQ ID NO: 44, a Phe        corresponding to position 76 of SEQ ID NO: 44, a Val        corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44, and the antibody        or fragment specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) amino acid sequence that        comprises a C-terminal domain comprising a mutation E670G in SEQ        ID NO:1, wherein the human comprises a nucleotide sequence        encoding said amino acid sequence and comprises an IGHG2*01        human heavy chain constant region gene segment. Optionally, the        antibody or fragment comprises an IGHG2*01 human heavy chain        constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 or E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human gamma-2 heavy        chain constant region that comprises a Pro corresponding to        position 72 of SEQ ID NO: 44, an Asn corresponding to position        75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ        ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44        and an Ala corresponding to position 257 of SEQ ID NO: 44 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment and (ii) a nucleotide sequence        encoding said proprotein convertase subtilisin/kexin type 9        (PCSK9) that comprises a C-terminal domain comprising said        mutation I474V or E670G in SEQ ID NO: 1.-   2. The antibody or antibody fragment of paragraph 1, wherein the    antibody comprises a human gamma-2 heavy chain constant region that    comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an    Asn corresponding to position 75 of SEQ ID NO: 44, a Phe    corresponding to position 76 of SEQ ID NO: 44, a Val corresponding    to position 161 of SEQ ID NO: 44 and an Ala corresponding to    position 257 of SEQ ID NO: 44.-   3. The antibody or antibody fragment of paragraph 1 or 2, wherein    the antibody comprises an IGHG2*01 human heavy chain constant    region.-   4. The antibody or antibody fragment of any one of paragraphs 1 to    3, wherein the human has been determined to comprise the nucleotide    sequence that encodes a PCSK9 comprising a C-terminal domain    comprising said mutation I474V or E670G in SEQ ID NO: 1 and/or a    proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein encoded by the nucleotide sequence of SEQ ID NO: 29 or 30.-   5. The antibody or antibody fragment of any one of paragraphs 1 to    4, the method comprising the step of determining that the human    comprises the nucleotide sequence that encodes a PCSK9 comprising a    C-terminal domain comprising said mutation I474V or E670G and/or a    proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein comprising said mutation I474V or E670G, optionally, wherein    the determining step is performed before administration of the    antibody to the human.-   6. The antibody or antibody fragment of paragraph 5, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation I474V or E670G in SEQ    ID NO: 1.-   7. The antibody or antibody fragment of paragraph 6, wherein the    assaying comprises contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   8. The antibody or antibody fragment of paragraph 6 or 7, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   9. The antibody or antibody fragment of any one of paragraphs 1 to    8, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 1 to    9, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   11. The antibody or antibody fragment of paragraph 9 or 10, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   12. The antibody or antibody fragment of any one of paragraphs 6 to    8, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a mutation    I474V or E670G, optionally, wherein said human is further indicated    as comprising the nucleotide sequence of SEQ ID NO: 28, or said    human is indicated as homozygous for a nucleotide sequence encoding    the PCSK9 C-terminal domain comprising a mutation I474V or E670G in    SEQ ID NO: 1.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication and high blood pressure.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   16. The antibody or antibody fragment of any one of paragraphs 1 to    15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   17. The antibody or antibody fragment of any one of paragraphs 1 to    16, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of reducing cholesterol level or maintaining previously    reduced cholesterol level in a human in need thereof, the method    comprising administering to said human an antibody or antibody    fragment that specifically binds a proprotein convertase    subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain    comprising a mutation I474 or E670G in SEQ ID NO: 1, wherein the    antibody or fragment comprises a human gamma-2 heavy chain constant    region that comprises an amino acid selected from the group    consisting of a Pro corresponding to position 72 of SEQ ID NO: 44,    an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe    corresponding to position 76 of SEQ ID NO: 44, a Val corresponding    to position 161 of SEQ ID NO: 44 and an Ala corresponding to    position 257 of SEQ ID NO: 44 and wherein said human comprises (i)    an IGHG2*01 human heavy chain constant region gene segment, or the    human expresses antibodies comprising human gamma-2 heavy chain    constant regions comprising such a Pro, Asn, Phe, Val and Ala    and (ii) a nucleotide sequence encoding said proprotein convertase    subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain    comprising said mutation I474V or E670G in SEQ ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human gamma-2 heavy chain        constant region that comprises a Pro corresponding to position        72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ        ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44,        a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44 and wherein said        human comprises (i) an IGHG2*01 human heavy chain constant        region gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation I474V in        SEQ ID NO: 1. Optionally, the antibody or fragment comprises an        IGHG2*01 human heavy chain constant region.    -   Another example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation E670G in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human gamma-2 heavy chain        constant region that comprises a Pro corresponding to position        72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ        ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44,        a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44 and wherein said        human comprises (i) an IGHG2*01 human heavy chain constant        region gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation E670G in        SEQ ID NO: 1. Optionally, the antibody or fragment comprises an        IGHG2*01 human heavy chain constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 or E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human gamma-2 heavy        chain constant region that comprises a Pro corresponding to        position 72 of SEQ ID NO: 44, an Asn corresponding to position        75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ        ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44        and an Ala corresponding to position 257 of SEQ ID NO: 44 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment and (ii) a nucleotide sequence        encoding said proprotein convertase subtilisin/kexin type 9        (PCSK9) that comprises a C-terminal domain comprising said        mutation I474V or E670G in SEQ ID NO: 1.-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the antibody comprises a    human gamma-1 heavy chain constant region that comprises a Pro    corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding    to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76    of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO:    44 and an Ala corresponding to position 257 of SEQ ID NO: 44.-   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises    an IGHG2*01 human heavy chain constant region.-   5. The method of any one of paragraphs 1 to 4, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation I474V    or E670G in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   6. The method of any one of paragraphs 1 to 5, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation I474V or E670G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   7. The method of paragraph 6, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation I474V or E670G in SEQ ID NO: 1.-   8. The method of paragraph 7, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   9. The method of paragraph 7 or 8, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   10. The method of any one of paragraphs 1 to 9, wherein said human    is or has been further determined to be substantially resistant to    statin treatment.-   11. The method of any one of paragraphs 1 to 10, wherein said human    is receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   12. The method of paragraph 10 or 11, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   13. The method of any one of paragraphs 7 to 9, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   14. The method of any one of paragraphs 1 to 13, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 28, or said human is indicated    as homozygous for a nucleotide sequence encoding the PCSK9    C-terminal domain comprising said mutation I474V or E670G in SEQ ID    NO: 1.-   15. The method of any one of paragraphs 1 to 14, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   17. The method of any one of paragraphs 1 to 16, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   18. The method of any one of paragraphs 1 to 17, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   1. An antibody or antibody fragment for use in a method of reducing    cholesterol level or maintaining previously reduced cholesterol    level in a human in need thereof, wherein the antibody or fragment    comprises a human kappa light chain constant region that comprises a    Val corresponding to position 84 of SEQ ID NO: 50 or a Cys    corresponding to position 87 of SEQ ID NO: 50, and the antibody or    fragment specifically binds a proprotein convertase subtilisin/kexin    type 9 (PCSK9) amino acid sequence that comprises a C-terminal    domain comprising a mutation I474V or E670G in SEQ ID NO:1, wherein    the human comprises a nucleotide sequence encoding said amino acid    sequence and comprises an IGKC*01 human light chain constant region    gene segment, or the human expresses antibodies comprising human    kappa light chain constant regions comprising such an Val and Cys.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   An antibody or antibody fragment for use in a method of reducing        cholesterol level or maintaining previously reduced cholesterol        level in a human in need thereof, wherein the antibody or        fragment comprises a human kappa light chain constant region        that comprises a Val corresponding to position 84 of SEQ ID NO:        50 and a Cys corresponding to position 87 of SEQ ID NO: 50, and        the antibody or fragment specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence        that comprises a C-terminal domain comprising a mutation I474V        in SEQ ID NO:1, wherein the human comprises a nucleotide        sequence encoding said amino acid sequence and comprises an        IGKC*01 human light chain constant region gene segment.        Optionally, the antibody or fragment comprises an IGKC*01 human        light chain constant region.    -   Another example provides:—    -   An antibody or antibody fragment for use in a method of reducing        cholesterol level or maintaining previously reduced cholesterol        level in a human in need thereof, wherein the antibody or        fragment comprises a human kappa light chain constant region        that comprises a Val corresponding to position 84 of SEQ ID NO:        50 and a Cys corresponding to position 87 of SEQ ID NO: 50, and        the antibody or fragment specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence        that comprises a C-terminal domain comprising a mutation E670G        in SEQ ID NO:1, wherein the human comprises a nucleotide        sequence encoding said amino acid sequence and comprises an        IGKC*01 human light chain constant region gene segment.        Optionally, the antibody or fragment comprises an IGKC*01 human        light chain constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 or E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human kappa light        chain constant region that comprises a Val corresponding to        position 84 of SEQ ID NO: 50 and a Cys corresponding to position        87 of SEQ ID NO: 50 and wherein said human comprises (i) an        IGKC*01 human heavy chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation I474V or E670G in SEQ ID NO: 1.-   2. The antibody or antibody fragment of paragraph 1, wherein the    antibody comprises a human kappa light chain constant region that    comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a    Cys corresponding to position 87 of SEQ ID NO: 50.-   3. The antibody or antibody fragment of paragraph 1 or 2, wherein    the antibody comprises an IGKC*01 human kappa chain constant region.-   4. The antibody or antibody fragment of any one of paragraphs 1 to    3, wherein the human has been determined to comprise the nucleotide    sequence that encodes a PCSK9 comprising a C-terminal domain    comprising said mutation I474V or E670G in SEQ ID NO: 1 and/or a    proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein encoded by the nucleotide sequence of SEQ ID NO: 29 or 30.-   5. The antibody or antibody fragment of any one of paragraphs 1 to    4, the method comprising the step of determining that the human    comprises the nucleotide sequence that encodes a PCSK9 comprising a    C-terminal domain comprising said mutation I474V or E670G and/or a    proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein comprising said mutation I474V or E670G, optionally, wherein    the determining step is performed before administration of the    antibody to the human.-   6. The antibody or antibody fragment of paragraph 5, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation I474V or E670G in SEQ    ID NO: 1.-   7. The antibody or antibody fragment of paragraph 6, wherein the    assaying comprises contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   8. The antibody or antibody fragment of paragraph 6 or 7, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   9. The antibody or antibody fragment of any one of paragraphs 1 to    8, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 1 to    9, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   11. The antibody or antibody fragment of paragraph 9 or 10, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   12. The antibody or antibody fragment of any one of paragraphs 6 to    10, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a mutation    I474V or E670G, optionally, wherein said human is further indicated    as comprising the nucleotide sequence of SEQ ID NO: 28, or said    human is indicated as homozygous for a nucleotide sequence encoding    the PCSK9 C-terminal domain comprising a mutation I474V or E670G in    SEQ ID NO: 1.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication and high blood pressure.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   16. The antibody or antibody fragment of any one of paragraphs 1 to    15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   17. The antibody or antibody fragment of any one of paragraphs 1 to    16, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of reducing cholesterol level or maintaining previously    reduced cholesterol level in a human in need thereof, the method    comprising administering to said human an antibody or antibody    fragment that specifically binds a proprotein convertase    subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain    comprising a mutation I474 or E670G in SEQ ID NO: 1, wherein the    antibody or fragment comprises a human kappa light chain constant    region that comprises a Val corresponding to position 84 of SEQ ID    NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50 and    wherein said human comprises (i) an IGKC*01 human light chain    constant region gene segment, or the human expresses antibodies    comprising human kappa light chain constant regions comprising such    an Val and Cys and (ii) a nucleotide sequence encoding said    proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises    a C-terminal domain comprising said mutation I474V or E670G in SEQ    ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human kappa light chain        constant region that comprises a Val corresponding to position        84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of        SEQ ID NO: 50 and wherein said human comprises (i) an IGKC*01        human light chain constant region gene segment and (ii) a        nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation I474V in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGKC*01 human        light chain constant region.    -   Another example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation E670G in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human kappa light chain        constant region that comprises a Val corresponding to position        84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of        SEQ ID NO: 50 and wherein said human comprises (i) an IGKC*01        human light chain constant region gene segment and (ii) a        nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation E670G in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGKC*01 human        light chain constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 or E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human kappa light        chain constant region that comprises a Val corresponding to        position 84 of SEQ ID NO: 50 and a Cys corresponding to position        87 of SEQ ID NO: 50 and wherein said human comprises (i) an        IGKC*01 human heavy chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation I474V or E670G in SEQ ID NO: 1.-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the antibody comprises a    human kappa light chain constant region that comprises a Val    corresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding    to position 87 of SEQ ID NO: 50.-   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises    an IGKC*01 human kappa chain constant region.-   5. The method of any one of paragraphs 1 to 4, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation I474V    or E670G in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   6. The method of any one of paragraphs 1 to 5, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation I474V or E670G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   7. The method of paragraph 6, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation I474V or E670G in SEQ ID NO: 1.-   8. The method of paragraph 7, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   9. The method of paragraph 7 or 8, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   10. The method of any one of paragraphs 1 to 9, wherein said human    is or has been further determined to be substantially resistant to    statin treatment.-   11. The method of any one of paragraphs 1 to 10, wherein said human    is receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   12. The method of paragraph 10 or 11, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   13. The method of any one of paragraphs 7 to 9, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   14. The method of any one of paragraphs 1 to 13, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 28, or said human is indicated    as homozygous for a nucleotide sequence encoding the PCSK9    C-terminal domain comprising said mutation I474V or E670G in SEQ ID    NO: 1.-   15. The method of any one of paragraphs 1 to 14, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   17. The method of any one of paragraphs 1 to 16, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   18. The method of any one of paragraphs 1 to 17, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-15 as follows.

-   1. An antibody or antibody fragment for use in a method of reducing    cholesterol level or maintaining previously reduced cholesterol    level in a human in need thereof, wherein the antibody or fragment    comprises a human IGLC2*01 lambda light chain constant region, and    the antibody or fragment specifically binds a proprotein convertase    subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a    C-terminal domain comprising a mutation I474V or E670G in SEQ ID    NO:1, wherein the human comprises a nucleotide sequence encoding    said amino acid sequence and comprises a human IGLC2*01 lambda light    chain constant region gene segment, or the human expresses    antibodies comprising human IGLC2*01 lambda light chain constant    regions.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   An antibody or antibody fragment for use in a method of reducing        cholesterol level or maintaining previously reduced cholesterol        level in a human in need thereof, wherein the antibody or        fragment comprises a human IGLC2*01 lambda light chain constant        region, and the antibody or fragment specifically binds a        proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid        sequence that comprises a C-terminal domain comprising a        mutation I474V in SEQ ID NO:1, wherein the human comprises a        nucleotide sequence encoding said amino acid sequence and        comprises a human IGLC2*01 lambda light chain constant region        gene segment. Optionally, the antibody or fragment comprises a        human IGLC2*01 lambda light chain constant region.    -   Another example provides:—    -   An antibody or antibody fragment for use in a method of reducing        cholesterol level or maintaining previously reduced cholesterol        level in a human in need thereof, wherein the antibody or        fragment comprises a human IGLC2*01 lambda light chain constant        region, and the antibody or fragment specifically binds a        proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid        sequence that comprises a C-terminal domain comprising a        mutation E670G in SEQ ID NO:1, wherein the human comprises a        nucleotide sequence encoding said amino acid sequence and        comprises a human IGLC2*01 lambda light chain constant region        gene segment. Optionally, the antibody or fragment comprises a        human IGLC2*01 lambda light chain constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 or E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human IGLC2*01        lambda light chain constant region and wherein said human        comprises (i) an IGLC2*01 human heavy chain constant region gene        segment and (ii) a nucleotide sequence encoding said proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising said mutation I474V or E670G in SEQ        ID NO: 1.-   2. The antibody or antibody fragment of paragraph 1, wherein the    human has been determined to comprise the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G in SEQ ID NO: 1 and/or a proprotein    convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded    by the nucleotide sequence of SEQ ID NO: 29 or 30.-   3. The antibody or antibody fragment of paragraphs 1 or 2, the    method comprising the step of determining that the human comprises    the nucleotide sequence that encodes a PCSK9 comprising a C-terminal    domain comprising said mutation I474V or E670G and/or a proprotein    convertase subtilisin/kexin type 9 (PCSK9) variant protein    comprising said mutation I474V or E670G, optionally, wherein the    determining step is performed before administration of the antibody    to the human.-   4. The antibody or antibody fragment of paragraph 3, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation I474V or E670G in SEQ    ID NO: 1.-   5. The antibody or antibody fragment of paragraph 4, wherein the    assaying comprises contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   6. The antibody or antibody fragment of paragraph 4 or 5, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   7. The antibody or antibody fragment of any one of paragraphs 1 to    6, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   8. The antibody or antibody fragment of any one of paragraphs 1 to    7, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   9. The antibody or antibody fragment of paragraph 7 or 8, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 4 to    8, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   11. The antibody or antibody fragment of any one of paragraphs 1 to    10, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a mutation    I474V or E670G, optionally, wherein said human is further indicated    as comprising the nucleotide sequence of SEQ ID NO: 28, or said    human is indicated as homozygous for a nucleotide sequence encoding    the PCSK9 C-terminal domain comprising a mutation I474V or E670G in    SEQ ID NO: 1.-   12. The antibody or antibody fragment of any one of paragraphs 1 to    11, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication and high blood pressure.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.

Further exemplary methods are in the paragraphs 1-16 as follows.

-   1. A method of reducing cholesterol level or maintaining previously    reduced cholesterol level in a human in need thereof, the method    comprising administering to said human an antibody or antibody    fragment that specifically binds a proprotein convertase    subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain    comprising a mutation I474 or E670G in SEQ ID NO: 1, wherein the    antibody or fragment comprises a human IGLC2*01 lambda light chain    constant region and wherein said human comprises (i) an IGLC2*01    human light chain constant region gene segment, or the human    expresses antibodies comprising a human IGLC2*01 lambda light chain    constant regions and (ii) a nucleotide sequence encoding said    proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises    a C-terminal domain comprising said mutation I474V or E670G in SEQ    ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human IGLC2*01 lambda light        chain constant region and wherein said human comprises (i) an        IGLC2*01 human light chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation I474V in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGLC2*01 human        light chain constant region.    -   Another example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation E670G in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human IGLC2*01 lambda light        chain constant region and wherein said human comprises (i) an        IGLC2*01 human light chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation E670G in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGLC2*01 human        light chain constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 or E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human IGLC2*01        lambda light chain constant region and wherein said human        comprises (i) an IGLC2*01 human heavy chain constant region gene        segment and (ii) a nucleotide sequence encoding said proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising said mutation I474V or E670G in SEQ        ID NO: 1-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the human has been    determined to comprise the nucleotide sequence that encodes a PCSK9    comprising a C-terminal domain comprising said mutation I474V or    E670G in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   4. The method of any one of paragraphs 1 to 3, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation I474V or E670G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   5. The method of paragraph 4, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation I474V or E670G in SEQ ID NO: 1.-   6. The method of paragraph 5, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   7. The method of paragraph 5 or 6, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   8. The method of any one of paragraphs 1 to 7, wherein said human is    or has been further determined to be substantially resistant to    statin treatment.-   9. The method of any one of paragraphs 1 to 8, wherein said human is    receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   10. The method of paragraph 8 or 9, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   11. The method of any one of paragraphs 5 to 7, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   12. The method of any one of paragraphs 1 to 11, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 28, or said human is indicated    as homozygous for a nucleotide sequence encoding the PCSK9    C-terminal domain comprising said mutation I474V or E670G in SEQ ID    NO: 1.-   13. The method of any one of paragraphs 1 to 12, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   14. The method of any one of paragraphs 1 to 13, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   15. The method of any one of paragraphs 1 to 14, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-15 as follows.

-   1. An antibody or antibody fragment for use in a method of reducing    cholesterol level or maintaining previously reduced cholesterol    level in a human in need thereof, wherein the antibody or fragment    comprises a human variable domain that is derived from the    recombination of a human VH gene segment, a human D gene segment and    a human JH gene segment, wherein the VH gene segment is as defined    in any one of clauses 80 to 90, and the antibody or fragment    specifically binds a proprotein convertase subtilisin/kexin type 9    (PCSK9) amino acid sequence that comprises a C-terminal domain    comprising a mutation I474V or E670G in SEQ ID NO:1, wherein the    human comprises a nucleotide sequence encoding said amino acid    sequence, and comprises said VH gene segment or expresses antibodies    comprising VH domains that are derived from the recombination of    said human VH gene segment, a human D gene segment and a human JH    gene segment.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.-   2. The antibody or antibody fragment of paragraph 1, wherein the    human has been determined to comprise the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G in SEQ ID NO: 1 and/or a proprotein    convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded    by the nucleotide sequence of SEQ ID NO: 29 or 30.-   3. The antibody or antibody fragment of paragraphs 1 or 2, the    method comprising the step of determining that the human comprises    the nucleotide sequence that encodes a PCSK9 comprising a C-terminal    domain comprising said mutation I474V or E670G and/or a proprotein    convertase subtilisin/kexin type 9 (PCSK9) variant protein    comprising said mutation I474V or E670G, optionally, wherein the    determining step is performed before administration of the antibody    to the human.-   4. The antibody or antibody fragment of paragraph 3, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation I474V or E670G in SEQ    ID NO: 1.-   5. The antibody or antibody fragment of paragraph 4, wherein the    assaying comprises contacting the biological sample with a. at least    one oligonucleotide probe comprising a sequence of at least 10    contiguous nucleotides that can specifically hybridize to and    identify in the biological sample a nucleotide sequence encoding the    PCSK9 that comprises the C-terminal domain comprising the mutation    I474V or E670G in SEQ ID NO: 1 or that specifically hybridizes to an    antisense of said sequence, wherein said nucleic acid hybridizes to    at least one nucleotide present in said selected sequence which is    not present in SEQ ID NO: 28 or hybridizes to an antisense sequence    thereby forming a complex when at least one nucleotide sequence    encoding the PCSK9 that comprises the C-terminal domain comprising    the mutation I474V or E670G in SEQ ID NO: 1 is present; and/or b. at    least one oligonucleotide probe comprising a sequence of at least 10    contiguous nucleotides of a nucleotide sequence encoding the PCSK9    that comprises the C-terminal domain comprising the mutation I474V    or E670G in SEQ ID NO: 1 or comprising an antisense sequence of said    contiguous nucleotides, wherein said sequence of contiguous    nucleotides comprises at least one nucleotide present in said    selected sequence which is not present in SEQ ID NO: 28 thereby    forming a complex when the nucleotide sequence encoding the PCSK9    that comprises a C-terminal domain comprising the mutation I474V or    E670G in SEQ ID NO: 1 is present; and detecting the presence or    absence of the complex, wherein detecting the presence of the    complex determines that the human comprises the PCSK9 that comprises    the C-terminal domain comprising the mutation I474V or E670G in SEQ    ID NO: 1.-   6. The antibody or antibody fragment of paragraph 4 or 5, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   7. The antibody or antibody fragment of any one of paragraphs 1 to    6, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   8. The antibody or antibody fragment of any one of paragraphs 1 to    7, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   9. The antibody or antibody fragment of paragraph 7 or 8, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 4 to    8, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   11. The antibody or antibody fragment of any one of paragraphs 1 to    10, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a mutation    I474V or E670G, optionally, wherein said human is further indicated    as comprising the nucleotide sequence of SEQ ID NO: 28, or said    human is indicated as homozygous for a nucleotide sequence encoding    the PCSK9 C-terminal domain comprising a mutation I474V or E670G in    SEQ ID NO: 1.-   12. The antibody or antibody fragment of any one of paragraphs 1 to    11, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication and high blood pressure.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.    Further exemplary methods are in the paragraphs 1-16 as follows.-   1. A method of reducing cholesterol level or maintaining previously    reduced cholesterol level in a human in need thereof, the method    comprising administering to said human an antibody or antibody    fragment that specifically binds a proprotein convertase    subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain    comprising a mutation I474 or E670G in SEQ ID NO: 1, wherein the    antibody or fragment comprises a human variable domain that is    derived from the recombination of a human VH gene segment, a human D    gene segment and a human JH gene segment, wherein the VH gene    segment is as defined in any one of clauses 80 to 90 and wherein    said human comprises (i) comprises said VH gene segment or expresses    antibodies comprising VH domains that are derived from the    recombination of said human VH gene segment, a human D gene segment    and a human JH gene segment and (ii) a nucleotide sequence encoding    said proprotein convertase subtilisin/kexin type 9 (PCSK9) that    comprises a C-terminal domain comprising said mutation I474V or    E670G in SEQ ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the human has been    determined to comprise the nucleotide sequence that encodes a PCSK9    comprising a C-terminal domain comprising said mutation I474V or    E670G in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   4. The method of any one of paragraphs 1 to 3, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation I474V or E670G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   5. The method of paragraph 4, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation I474V or E670G in SEQ ID NO: 1.-   6. The method of paragraph 5, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   7. The method of paragraph 5 or 6, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   8. The method of any one of paragraphs 1 to 7, wherein said human is    or has been further determined to be substantially resistant to    statin treatment.-   9. The method of any one of paragraphs 1 to 8, wherein said human is    receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   10. The method of paragraph 8 or 9, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   11. The method of any one of paragraphs 5 to 7, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   12. The method of any one of paragraphs 1 to 11, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 28, or said human is indicated    as homozygous for a nucleotide sequence encoding the PCSK9    C-terminal domain comprising said mutation I474V or E670G in SEQ ID    NO: 1.-   13. The method of any one of paragraphs 1 to 12, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   14. The method of any one of paragraphs 1 to 13, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   15. The method of any one of paragraphs 1 to 14, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Regimens

A: The invention further provides the following regimens, ligands andkits.

-   1. A method for treating a human PCSK9-mediated disease or condition    in a human by targeting a rare variant human PCSK9, the method    comprising administering to the human a ligand (eg, an antibody or    fragment) that has been determined to specifically bind to said    PCSK9 variant; wherein the human expresses said PCSK9 variant or the    genome of the human comprises a nucleotide sequence encoding said    PCSK9 variant; wherein said human is treated for said disease or    condition.    The variant PCSK9 can, for example, be any rare variant as described    herein.    For example, there is provided:    1a. A method for treating a PCSK9-mediated disease or condition in a    human by targeting a PCSK9 that comprises a C-terminal domain amino    acid polymorphism (compared to SEQ ID NO: 1), the method comprising    administering to the human a ligand (eg, an antibody or fragment)    that has been determined to specifically bind to a PCSK9 comprising    a C-terminal domain comprising I474V or 670G (numbering according to    SEQ ID NO:1); wherein the human expresses said PCSK9 or the genome    of the human comprises a nucleotide sequence encoding said PCSK9;    wherein said human is treated for said disease or condition.    In an embodiment, determination of said specific binding is by    reference to binding assay data, eg, as determined using SPR or    ELISA. Determination may, for example, be by reference to    information in a printed publication, eg, with knowledge of data    presented in the present or another patent application or in a    journal article. Once armed with such knowledge (eg, in the absence    of further testing of binding), the skilled person is able—by    direction of the present invention—to treat a relevant human whose    genotype or phenotype matches the binding specificity of the ligand.    The antibody or fragment can be according to any configuration,    example, embodiment, aspect, clause or paragraph herein.    In an embodiment, the method comprises, before said administering,    selecting a human comprising said nucleotide sequence encoding the    PCSK9, wherein the human is said human in clause 1 (eg, la).-   2. The method of clause 1, comprising before said administering the    step of determining that the ligand specifically binds to said    PCSK9, eg, using SPR or ELISA.-   3. The method of clause 1 or 2, wherein the specific binding to said    PCSK9 is binding with a dissociation constant (Kd) of 1 nM or less,    eg, 100, 10 or 1 pM or less.-   4. The method of any of clauses 1 to 3 (eg, clause la), wherein the    condition is elevated LDL-cholesterol or is caused by elevated    LDL-cholesterol.-   5. The method of clause 4 (eg, when dependent from clause la),    wherein the condition is selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   6. The method of any one of clauses 1 to 5 (eg, when dependent from    clause la), wherein the human has been determined to comprise the    nucleotide sequence that encodes a PCSK9 comprising a C-terminal    domain comprising said mutation I474V or E670G in SEQ ID NO: 1    and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)    variant protein encoded by the nucleotide sequence of SEQ ID NO: 29    or 30.-   7. The method of any one of clauses 1 to 6 (eg, when dependent from    clause la), comprising the step of determining that the human    comprises the nucleotide sequence that encodes a PCSK9 comprising a    C-terminal domain comprising said mutation I474V or E670G and/or a    proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein comprising said mutation I474V or E670G, optionally, wherein    the determining step is performed before administration of the    antibody to the human.-   8. The method of clause 7, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding the PCSK9 that comprises the C-terminal domain    comprising the mutation I474V or E670G in SEQ ID NO: 1.-   9. The method of clause 8, wherein the assaying comprises contacting    the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   10. The method of clause 8 or 9, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   11. The method of any one of clauses 1 to 10, wherein said human is    or has been further determined to be substantially resistant to    statin treatment.-   12. The method of any one of clauses 1 to 11, wherein said human is    receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   13. The method of clauses 11 or 12, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   14. The method of any one of clauses 8 to 10, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   15. The method of any one of clauses 1 to 14, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 28, or said human is indicated    as homozygous for a nucleotide sequence encoding the PCSK9    C-terminal domain comprising said mutation I474V or E670G in SEQ ID    NO: 1.-   16. The method of any one of clauses 1 to 15, wherein said human has    been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   17. The method of any one of clauses 1 to 16, wherein said antibody    or antibody fragment treats or reduces the risk in said human of at    least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   18. The method of any one of clauses 1 to 17, wherein the nucleotide    sequence is SEQ ID NO: 29 or 30.-   19. The method of any one of clauses 1 to 18, wherein said antibody    or antibody fragment is administered by intravenous or subcutaneous    administration and/or is comprised in an injectable preparation.-   20. A ligand (eg, an antibody or fragment) for use in the method of    any one of clauses 1 to 19, wherein the ligand specifically binds    the PCSK9.-   21. A kit comprising the ligand of clause 20 and instructions for    carrying out the method of any one of clauses 1 to 19.

B: The invention further provides the following regimens, ligands andkits.

-   1. A method of reducing cholesterol level or maintaining a    previously reduced cholesterol level in a human in need thereof, the    method comprising:—    -   a. Carrying out an initial treatment of said human for an        initial treatment period by administering an anti-human PCSK9        ligand (eg, an antibody or fragment) to said human, wherein (i)        the ligand has been determined to specifically bind to a PCSK9        comprising a C-terminal domain comprising I474V or 670G        (numbering according to SEQ ID NO:1); (ii) the human expresses        said PCSK9 or the genome of the human comprises a nucleotide        sequence encoding said PCSK9 and (iii) the human has received or        is receiving statin treatment to lower or maintain cholesterol        level; wherein the initial treatment comprises the        administration of a single or multiple doses of the ligand to        the human;    -   b. Determining to (i) terminate statin treatment (ii) keep the        human off statin treatment; or (iii) reduce statin treatment        after said initial treatment period; and    -   c. Continuing to administer the ligand to said patient after        said time period has expired, thereby reducing cholesterol level        or maintaining a previously reduced cholesterol level in said        human.        In an embodiment, determination of said specific binding is by        reference to binding assay data, eg, as determined using SPR or        ELISA. Determination may, for example, be by reference to        information in a printed publication, eg, with knowledge of data        presented in the present or another patent application or in a        journal article. Once armed with such knowledge (eg, in the        absence of further testing of binding), the skilled person is        able—by direction of the present invention—to treat a relevant        human whose genotype or phenotype matches the binding        specificity of the ligand.        The antibody or fragment can be according to any configuration,        example, embodiment, aspect, clause or paragraph herein.        A pharmaceutically-effective amount of said ligand is        administered.        In an embodiment, the method comprises, before said        administering, selecting a human comprising said nucleotide        sequence encoding the PCSK9, wherein the human is said human        recited in clause 1.        In an example, the initial treatment period is 7 days, 14 days,        21 days, 28 days, a month, two months, three months, four        months, five months, six months, seven months, eight months,        nine months or a year.-   2. The method of clause 1, wherein the human has or is suffering    from statin-associated memory loss or a statin-associated    neurodegenerative condition, or has or is at increased risk of    diabetes (eg, statin-associated diabetes).-   3. The method of clause 1 or 2, comprising, before said initial    treatment, the step of determining that the human has or is    suffering from statin-associated memory loss or a statin-associated    neurodegenerative condition, or has or is at increased risk of    diabetes (eg, statin-associated diabetes).-   4. The method of clause 2 or 3, comprising, after step (b) (eg,    during step (c)) determining that the memory loss or said    neurodegenerative condition has improved.-   5. The method of any one of clauses 1 to 4, wherein said human is    over 40 years of age (eg, 50 or over, 55 or over, 60 or over, 65 or    over, or 70 or over).-   6. The method of any one of clauses 1 to 5, wherein step (c)    comprises determining to increase the doses of said ligand to be    administered after said initial treatment period and administering    said increased doses to said human.-   7. The method of any one of clauses 1 to 6, wherein step (b)    comprises determining that the human is intolerant or refactory to    treatment by a statin.-   8. The method of any one of clauses 1 to 7, wherein the initial    treatment comprises the administration of a statin, fenofibrate (eg,    Tricor™ or Lofibra™) or ezetimibe to the human in addition to the    ligand.-   9. The method of any one of clauses 1 to 8, wherein step (b)    comprises terminating or reducing statin, fenofibrate (eg, Tricor™    or Lofibra™) or ezetimibe treatment during step (c).-   10. The method of any one of clauses 1 to 9, comprising increasing    (ie, increasing compared to the initial treatment dose) the ligand    dose during step (c).-   11. The method of any one of clauses 1 to 10, wherein the human has    received high dose statin treatment prior to the initial treatment,    and wherein step (c) comprises administering a lower (eg, a medium    or low) dose statin treatment in addition to said ligand.    -   The skilled person is familiar with the meaning of high, medium        and low dose treatments (and how to determine according to each        patient, eg, the patient's body mass). For example, the statin        is selected from rosuvastatin, atorvastatin and simvastatin.    -   For example daily statin doses are as follows:—Low 10 to 20 mg        (eg, 10 mg); medium >20 and <60 mg (eg, 40 mg); high 60-100 mg        (eg, 80 mg).-   12. The method of any one of clauses 1 to 10, wherein the human has    received medium dose statin treatment prior to the initial    treatment, and wherein step (c) comprises administering a lower (eg,    a low) dose statin treatment or no statin in addition to said    ligand.-   13. The method of any one of clauses 1 to 10, wherein the human has    received low dose statin treatment prior to the initial treatment,    and wherein step (c) comprises administering no statin in addition    to said ligand.-   14. The method of any one of clauses 1 to 13, comprising, before the    initial treatment, the step of determining that the ligand    specifically binds to said PCSK9, eg, using SPR or ELISA.-   15. The method of any one of clauses 1 to 14, wherein the specific    binding to said PCSK9 is binding with a dissociation constant (Kd)    of 1 nM or less, eg, 100, 10 or 1 pM or less.-   16. The method of any of clauses 1 to 15, wherein the human is at    risk of or suffering from a condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   17. The method of clause 16, wherein step (c) treats or reduces the    risk of said condition in the human.-   18. The method of any one of clauses 1 to 17, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation I474V    or E670G in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   19. The method of any one of clauses 1 to 18, comprising the step of    determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation I474V or E670G and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation I474V or E670G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   20. The method of clause 19, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation I474V or E670G in SEQ ID NO: 1.-   21. The method of clause 20, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or that        specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        I474V or E670G in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation I474V or E670G in SEQ        ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation I474V or E670G in SEQ ID NO: 1.-   22. The method of clause 20 or 21, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   23. The method of any one of clauses 1 to 22, wherein said human is    or has been further determined to be substantially resistant to    statin treatment.-   24. The method of any one of clauses 20 to 22, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   25. The method of any one of clauses 1 to 24, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 28, or said human is indicated    as homozygous for a nucleotide sequence encoding the PCSK9    C-terminal domain comprising said mutation I474V or E670G in SEQ ID    NO: 1.-   26. The method of any one of clauses 1 to 25, wherein said human has    been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    and high blood pressure.-   27. The method of any one of clauses 1 to 26, wherein the nucleotide    sequence is SEQ ID NO: 29 or 30.-   28. The method of any one of clauses 1 to 27, wherein said ligand    (eg, antibody or antibody fragment) is administered by intravenous    or subcutaneous administration and/or is comprised in an injectable    preparation.-   29. A ligand (eg, an antibody or fragment) for use in the method of    any one of clauses 1 to 28, wherein the ligand specifically binds    the PCSK9.-   30. A kit comprising the ligand of clause 29 and instructions for    carrying out the method of any one of clauses 1 to 28.

In an example of any aspect of the invention, the ligand (eg, antibodyor fragment, eg, alirocumab, bocovizumab or evolocumab, or an antibodyor fragment comprising the variable domains of 316P or 31H4) isadministered to the human at a two-weekly dose of from 75 to 150 mg (eg,from 75 to 150 mg administered once or twice over a two-week period). Inan example, the ligand is for such administration to the human.

Determination of Specific Binding of Ligands of the Invention to PCSK9Variants

Method of SPR Determination of Binding

Binding of the antibodies to the PCSK9 variants was carried out by SPRusing the ProteOn XPR36™ Array system (BioRad). An anti-human IgGsurface (Jackson Labs 109-005-008) was created on a GLC Biosensor chipby primary amine coupling. Test antibodies were captured on this surfaceas ligands. The PCSK9 variants were used as analytes and passed over thecaptured antibodies at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. Bindingcurves were double referenced using a buffer injection (i.e. 0 nM) toremove baseline drift and injection artefacts. Regeneration of thecapture surface was with 100 mM phosphoric acid which removed thecaptured antibody allowing another cycle of capture and binding. Thebinding sensorgrams generated were analysed using the 1:1 model inherentto the ProteOn XPR36 Array system analysis software. The assay wasperformed at 25° C. and using 1×HBS-EP (Teknova) as running buffer.

Data

Three antibodies were tested and the resulting binding data arepresented below (Table 3). Antibodies 316P and 31H4 are antibodiesdisclosed in US20110065902A1 (the sequences of these antibodies andtheir variable domains are incorporated herein by reference for possibleuse in the present invention and possible inclusion in claims herein).Antibody 316P comprises heavy chain variable domains derived fromrecombination of human VH3-23*04 and JH2*01 (with a D), and light chainvariable domains derived from recombination of human Vκ4-1*01 andJκ2*01.

Evolocumab comprises a human IGHG2*01 heavy chain and a human IGLC2*01lambda light chain, a VH derived from recombination of human IGHV1-18*01and IGHJ6*01 (with a D segment) and a Vλ derived from recombination ofhuman IGLV2-14*01 and IGLJ2*01.

TABLE 3 SPR Determination of Ligand Binding Specificity for PCSK9Variants Variant/ Antibody ka (1/Ms) kd (1/s) KD (nM) PCSK9 a 316P1.37E+06 2.75E−04 0.201 31H4 1.14E+06 6.38E−05 0.056 Evolocumab 1.14E+052.62E−05 0.229 PCSK9 a′ 316P 1.50E+06 2.72E−04 0.181 31H4 1.23E+066.06E−05 0.049 Evolocumab 1.24E+05 2.29E−05 0.185 PCSK9 c 316P 1.49E+062.75E−04 0.184 31H4 1.22E+06 5.69E−05 0.047 Evolocumab 1.20E+05 2.20E−050.183 PCSK9 r 316P 1.40E+06 2.76E−04 0.197 31H4 1.15E+06 5.82E−05 0.051Evolocumab 1.16E+05 2.67E−05 0.230 PCSK9 f 316P 1.39E+06 2.82E−04 0.20331H4 1.13E+06 5.95E−05 0.053 Evolocumab 1.16E+05 2.66E−05 0.229 PCSK9 p316P 1.39E+06 2.73E−04 0.196 31H4 1.14E+06 6.12E−05 0.054 Evolocumab1.14E+05 2.50E−05 0.219

Results

The results showed that all antibodies tested bound to PCSK9 variantsequally, with any binding variation seen being within experimental errorfor such a strong affinity interaction.

Thus, the invention determines that an antibody with the followingprofile can specifically bind one or more variants of the invention:—

1. An antibody (eg, 316P or alirocumab) that comprises heavy chainvariable domains derived from recombination of human IGHV3-23*04 andIGHJH2*01 (with a D), and light chain variable domains derived fromrecombination of human IGKV4-1*01 and IGKJ2*01; or2. An antibody (eg, evolocumab) that comprises human heavy chainvariable domains derived from recombination of human IGHV1-18*01 andIGHJ6*01 (with a D segment) and light chain variable domains derivedfrom recombination of human IGLV2-14*01 and IGLJ2*01.

Thus, according to the invention, the skilled person is hereby providedwith the required determination of specific binding of the ligand toPCSK9 variants. Applications of this determination are set out above inthe context of methods and other aspects of the invention.

REFERENCES

-   Horton et al, Trends Biochem Sci. 2007 February; 32(2):71-7. Epub    2007 Jan. 9, Molecular biology of PCSK9: its role in LDL metabolism.-   Seidah and Prat, J Mol Med (Berl). 2007 July; 85(7):685-96. Epub    2007 Mar. 10, The proprotein convertases are potential targets in    the treatment of dyslipidemia.-   Benjannet et al, J Biol Chem. 2004 Nov. 19; 279(47):48865-75. Epub    2004 Sep. 9, NARC-1/PCSK9 and its natural mutants: zymogen cleavage    and effects on the low density lipoprotein (LDL) receptor and LDL    cholesterol.-   Lagace et al, J Clin Invest. 2006 November; 116(11):2995-3005,    Secreted PCSK9 decreases the number of LDL receptors in hepatocytes    and in livers of parabiotic mice.-   Maxwell et al, Proc Natl Acad Sci USA. 2005 Feb. 8; 102(6):2069-74.    Epub 2005 Jan. 27, Overexpression of PCSK9 accelerates the    degradation of the LDLR in a post-endoplasmic reticulum compartment.-   Park et al, J Biol Chem. 2004 Nov. 26; 279(48):50630-8. Epub 2004    Sep. 22, Post-transcriptional regulation of low density lipoprotein    receptor protein by proprotein convertase subtilisin/kexin type 9a    in mouse liver.-   Rashid et al, Proc Natl Acad Sci USA. 2005 Apr. 12; 102(15):5374-9.    Epub 2005 Apr. 1, Decreased plasma cholesterol and hypersensitivity    to statins in mice lacking Pcsk9.-   Kotowski et al, Am J Hum Genet. 2006 March; 78(3):410-22. Epub 2006    Jan. 20, A spectrum of PCSK9 alleles contributes to plasma levels of    low-density lipoprotein cholesterol.-   Chen et al, J Am Coll Cardiol. 2005 May 17; 45(10):1611-9. Epub 2005    Apr. 21, A common PCSK9 haplotype, encompassing the E670G coding    single nucleotide polymorphism, is a novel genetic marker for plasma    low-density lipoprotein cholesterol levels and severity of coronary    atherosclerosis.-   Pisciotta et al, Atherosclerosis. 2006 June; 186(2):433-40. Epub    2005 Sep. 23, Additive effect of mutations in LDLR and PCSK9 genes    on the phenotype of familial hypercholesterolemia.-   Zhao et al, Am J Hum Genet. 2006 September; 79(3):514-23. Epub 2006    Jul. 18, Molecular characterization of loss-of-function mutations in    PCSK9 and identification of a compound heterozygote.-   Seidah et al, Proc Natl Acad Sci USA. 2003 Feb. 4; 100(3):928-33.    Epub 2003 Jan. 27, The secretory proprotein convertase neural    apoptosis-regulated convertase 1 (NARC-1): liver regeneration and    neuronal differentiation.

TABLE 4 1000 GENOMES PROJECT HUMAN POPULATIONS Population Europeanancestry Utah residents (CEPH) with Northern and Western Europeanancestry (CEU) Toscani in Italia (TSI) British from England and Scotland(GBR) Finnish from Finland (FIN) Iberian populations in Spain (IBS) EastAsian ancestry Han Chinese in Beijing, China (CHB) Japanese in Toyko,Japan (JPT) Han Chinese South (CHS) Chinese Dai in Xishuangbanna (CDX)Kinh in Ho Chi Minh City, Vietnam (KHV) Chinese in Denver, Colorado(CHD) (pilot 3 only) West African ancestry Yoruba in Ibadan, Nigeria(YRI) Luhya in Webuye, Kenya (LWK) Gambian in Western Division, TheGambia (GWD) Malawian in Blantyre, Malawi (MAB) West African Population(TBD) Americas African Ancestry in Southwest US (ASW) African Americanin Jackson, MS (AJM) African Caribbean in Barbados (ACB) MexicanAncestry in Los Angeles, CA (MXL) Puerto Rican in Puerto Rico (PUR)Colombian in Medellin, Colombia (CLM) Peruvian in Lima, Peru (PEL) SouthAsian ancestry Ahom in the State of Assam, India Kayadtha in Calcutta,India Reddy in Hyderabad, India Maratha in Bombay, India Punjabi inLahore, PakistanBelow is a summary of the ethnic populations as per the 1000 GenomesProject sequences.

TABLE 5 TOIs & Related Diseases, Conditions and Example Ligands ExampleDisease or Human TOI Condition Example Ligand Il-17a InflammatoryDisease AIN457 Uveitis Ixekizumab Rheumatoid Arthritis PsoriasisAngiotensin II Receptor Type 1 (AT₁) Hypertension LCZ696 NeprilysinHypertension LCZ696 Metabotropic Glutamate Receptor 5 (Mglur5) Fragile XSyndrome AFQ056 Mavoglurant A Histone Deacetylase Cancer, Eg, MultipleLBH589 Myeloma Panobinostat Diacylglycerol acyltransferase-1 (DGAT-1)Familial LCQ908 Chylomicronaemia Pradigastat Syndrome Smoothened (Smo)Basal cell carcinoma LDE225 Solid tumour Myelofibrosis MedulloblastomaSolid tumour Smoothened (Smo) receptor Basal cell carcinoma LEQ506 Solidtumors ALK Non small cell lung LDK378 carcinoma (NSCLC)phosphatidylinositol-3-kinase (PI3K) Cancer, eg, solid tumour, BKM120mTOR breast cancer, Renal cell AKT carcinoma (RCC), Endometrial cancer,Non- small cell lung cancer (NSCLC), prostate cancer, Glioblastomamultiforme (GBM) CRPC GIST Myelofibrosis mTOR Cancer, eg, solid tumour,BEZ235 PI3K breast cancer, Renal cell AKT carcinoma (RCC), Endometrialcancer, Non- small cell lung cancer (NSCLC), prostate cancer,Glioblastoma multiforme (GBM) CRPC GIST Myelofibrosis PI3Kα Cancer, eg,solid tumour, BYL719 mTOR breast cancer, Renal cell PI3K carcinoma(RCC), AKT Endometrial cancer, Non- small cell lung cancer (NSCLC),prostate cancer, Glioblastoma multiforme (GBM) CRPC GIST MyelofibrosisMitogen-activated ERK kinase 1 (MEK1) Cancer, eg, solid tumour, MEK162Mitogen-activated ERK kinase 2 (MEK2) melanoma, pancreatic, colon, lungor thyroid cancer Metastasis protein kinase Acute myeloid leukemiaPKC412 protein kinase C (AML) Midostaurin FLT-3 Myelodysplastic syndromec-KIT (MDS) Aggressive systemic mastocytosis (ASM) ActRIIB Sporadicinclusion body BYM338 myositis (sIBM) bimagrumab CD19 Cancer, eg, CTL019(formerly CART- Leukaemia, mast 19) cell leukemia, Acute LymphoblasticLeukemia, Chronic lymphocytic leukemia (CLL) or Hematological tumors11β-hydroxylase Cushing's syndrome LCI699 BRAF Cancer, eg, melanomaLGX818 Receptor Tyrosine Kinase Cancer, eg, solid TKI258 (formerly CHIR-FGFR tumour 258) Breast Cancer Dovitinib Endometrial cancerHepatocellular carcinoma Renal cell carcinoma (RCC) Bladder cancermultiple myeloma (MM), hepatocellular carcinoma endometrial cancer DACenzyme hematologic malignancy LBH589 Multiple myeloma panobinostatMyel\odysplastic syndrome (MDS) Myelofibrosis HSP90 Cancer, eg, BreastAUY922 Cancer, gastric cancer or Non- small cell lung cancer (NSCLC)FGFR Cancer, eg, solid BGJ398 tumour Breast Cancer Endometrial cancerHepatocellular carcinoma Renal cell carcinoma (RCC) Bladder cancermultiple myeloma (MM), hepatocellular carcinoma endometrial cancer cIAP1Cancer, eg, solid LCL161 cIAP2 tumour Breast Cancer Endometrial cancerHepatocellular carcinoma Renal cell carcinoma (RCC) Bladder cancermultiple myeloma (MM), hepatocellular carcinoma endometrial cancer AktCancer, eg, Solid GDC-0068 tumour, gastric RG7440 cancer (eg,castration-resistant prostate cancer), prostate cancer, gastroesophagealjunction cancer CD22 Hematologic malignancies DCDT2980S Non-Hodgkin'slymphoma RG7593 (eg, relapsed or refractory follicular non-Hodgkin'slymphoma) Diffuse large B-cell lymphoma (eg, relapsed or refractorydiffuse large B- cell lymphoma) CD79b Hematologic malignancies DCDS4501ANon-Hodgkin's lymphoma RG7596 (eg, relapsed or refractory follicularnon-Hodgkin's lymphoma) Diffuse large B-cell lymphoma (eg, relapsed orrefractory diffuse large B- cell lymphoma) endothelin B receptor (ETBR)Melanoma, eg, metastatic DEDN6526A or unresectable melanoma RG7636 HER3Cancer, eg, metastatic MEHD7945A epithelial tumour, RG7597 metastaticsquamous cell carcinoma of the head and neck cancer, metastaticcolorectal cancer EGFR Cancer, eg, metastatic MEHD7945A epithelialtumour, RG7597 metastatic squamous cell Necitumumab carcinoma of thehead and neck cancer, metastatic colorectal cancer MUC16 Cancer, eg,ovarian cancer DMUC5754A (eg, platinum-resistant RG7458 ovarian cancer)sodium-dependent phosphate transport protein Cancer, eg, metastatic non-DNIB0600A 2b (NaPi2b) squamous non-small cell RG7599 lung cancer,ovarian cancer (eg, platinum-resistant ovarian cancer) PDL1 (programmeddeath ligand 1) Cancer, eg, solid tumour MPDL3280A metastatic melanomaRG7446 non-small cell lung cancer STEAP1 (six-transmembrane epithelialCancer, eg, prostate cancer DSTP3086S antigen of the prostate 1) (eg,metastatic castration- RG7450 resistant prostate cancer) Bcl-2 Cancer,eg, leukaemia (eg, GDC-0199 chronic lymphocytic RG7601 leukaemia),non-Hodgkin's lymphoma Checkpoint kinase 1 (ChK1) Cancer, eg, solidtumour, GDC-0425 refractory solid tumour or RG7602 lymphoma GDC-0575RG7741 mitogen activated protein kinase kinase Cancer, eg, solid tumour,GDC-0973 (MAPKK) melanoma (eg, metastatic RG7421 MEK melanoma)Cobimetinib GDC-0623 RG7420 epidermal growth factor domain-like-7Cancer, eg, colorectal Parsatuzumab (EGFL7) cancer (eg, metastaticMEGF0444A colorectal cancer), NSCLC RG7414 phosphatidylinositol-3-kinase(PI3K) Cancer, eg, prostate cancer, GDC-0032 renal cell carcinoma,RG7604 endometrial cancer, breast cancer (eg, HER2-negative GDC-0084metastatic breast cancer, RG7666 metastatic hormone Pictilisibreceptor-positive breast GDC-0941 cancer), solid tumour RG7321 mTORCancer, eg, prostate cancer, GDC-0980 TORC1 renal cell carcinoma, RG7422TORC2 endometrial cancer, breast PI3K cancer (eg, HER2-negativemetastatic breast cancer, metastatic hormone receptor-positive breastcancer), solid tumour IL17 Autoimmune disease Secukinumab Ankylosingspondylitis; ixekizumab Asthma; Multiple myeloma; Multiple sclerosis;Polymyalgia rheumatica; Psoriasis; Psoriatic arthritis; Rheumatoidarthritis; Uveitis M1 prime segment of membrane IgE Allergic AsthmaQuilizumab MEMP1972A RG7449 IFN alpha Autoimmune disease RontalizumabSystemic lupus erythematosus PCSK9 (proprotein convertasesubtilisin/kexin coronary heart disease MPSK3169A type 9) (CHD) or highrisk of CHD RG7652 hyperlipidaemia hypercholesterolaemia strokeatherosclerosis a cardiovascular disease or condition a conditionassociated with elevated LDL Vascular Endothelial Growth Factor-ADiabetic Macular Edema EYLEA ® (VEGF-A) (DME), Aflibercept PlacentalGrowth Factor (PlGF) Branch Retinal Vein AVASTIN ™ Occlusion (BRVO)LUCENTIS ™ Wet age-related macular degeneration (Wet AMD) IL-6 receptorInflammatory disease, eg, Sarilumab rheumatoid arthritis REGN88 Uveitis(eg, non-infectious uveitis) Ankylosing spondylitis; Cancer PCSK9(proprotein convertase subtilisin/kexin coronary heart diseaseAlirocumab type 9) (CHD) or high risk of CHD REGN727 hyperlipidaemiaLY3015014 hypercholesterolaemia stroke atherosclerosis a cardiovasculardisease or condition a condition associated with elevated LDL NGFOsteoarthritis Fasinumab Pain REGN475 IL-4 receptor alpha Allergicasthma Dupilumab IL-13 receptor eosinophilic asthma REGN668 Atopicdermatitis delta-like ligand-4 (Dll4) Cancer Enoticumab REGN421angiopoietin-2 (Ang2) Cancer Nesvacumab REGN910 GDF8 Metabolic disorderREGN1033 cancer, obesity, diabetes, LY2495655 arthritis, multiplesclerosis, muscular dystrophy, amyotrophic lateral sclerosis,Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolicsyndromes (including, but not limited to diabetes, obesity, nutritionaldisorders, organ atrophy, chronic obstructive pulmonary disease andanorexia) Disuse muscle atrophy cancer-related cachexia ERBB3 CancerREGN1400 angiopoietin -1 (Ang1) Cancer, eg, ovarian cancer AMG386angiopoietin -1 (Ang2) VEGF receptor Cancer Motesanib PDGF receptorNSCLC AMG 706 Stem cell factor receptor Breast cancer Thyroid cancertype 1 insulin-like growth factor receptor Cancer Ganitumab (IGF1R)Breast cancer Linsitinib Pancreatic cancer ASP7487 hepatocyte growthfactor (HGF) Cancer Rilotumumab Breast cancer Pancreatic cancer HER3Cancer AMG 888 ErbB3 Breast cancer U3-1287 Pancreatic cancer IL-17receptor Inflammatory disease AMG 827 Asthma brodalumab Psoriasissclerostin Bone-related condition AMG 785 postmenopausal CDP7851osteoporosis fracture healing glucokinase Diabetes AMG 151 ARRY-403PCSK9 (proprotein convertase subtilisin/kexin coronary heart disease AMG145 type 9) (CHD) or high risk of CHD Evolocumab hyperlipidaemiahypercholesterolaemia stroke atherosclerosis a cardiovascular disease orcondition a condition associated with elevated LDL VLA 2 Inflammatorybowel SAR339658 Integrin α2β1 disease IL-4 Idiopathic pulmonarySAR156597 IL-13 fibrosis lysophosphatidic acid receptor Systemicsclerosis SAR100842 LPA-1 fibrosis LPA-3 Androgen receptor Cancer, eg,prostate cancer, MDV3100 breast cancer enzalutamide HER1 Cancer, eg,NSCLC Erlotinib EGFR ERBITUX ™ VECTIBIX ™ VEGF receptor 1 Cancer, eg,colorectal ASP4130 VEGF receptor 2 cancer, breast cancer tivozanib VEGFreceptor 3 JAK Inflammatory disease, eg, ASP015K JAK1 rheumatoidarthritis Baricitinib JAK2 Diabetic nephropathy CD40 Prevention of organASP1240 transplant rejection GnRH Endometriosis ASP1707 Cancer, eg,prostate cancer degarelix PDE9 Lower unirary tract ASP4901 symptomsassociated with benign prostatic hyperplasia TNF alpha Inflammatorydisease, eg, Certolizumab pegol rheumatoid arthritis, psoriasis,chrohn's disease, IBD Programmed cell death protein 1 Cancer NivolumabChronic myelocytic MK-3475 leukemia; Hepatitis C virus infection;Hepatocellular carcinoma; Hodgkins disease; Melanoma; Multiple myeloma;Non- Hodgkin lymphoma; Non- small-cell lung cancer; Renal cellcarcinoma; Solid tumor; Stage IV melanoma Hepatocyte growth factorCancer onartuzumab MET Glioblastoma; Hepatocellular carcinoma;Metastatic colorectal cancer; Metastatic non small cell lung cancer;Metastatic stomach cancer; Non-small-cell lung cancer Angiopoietinligand-1 Breast tumor; Cancer; trebananib Angiopoietin ligand-2Colorectal tumor; Fallopian Tek tyrosine kinase receptor tube cancer;Gastrointestinal tumor; Glioblastoma; Hepatocellular carcinoma;Metastatic esophageal cancer; Metastatic gastrointestinal cancer;Metastatic non small cell lung cancer; Metastatic ovary cancer;Metastatic renal cancer; Ovary tumor; Peritoneal tumor; Transitionalcell carcinoma CD37 modulator Cancer elotuzumab Lymphocyte functionantigen-3 receptor Multiple myeloma SLAM family member 7 IL-2 Multiplesclerosis daclizumab IL-2 receptor alpha EGFR Cancer necitumumabMetastatic non small cell lung cancer; Solid tumor IL-5 Asthma;Eosinophilic reslizumab esophagitis B-lymphocyte cell adhesion moleculeCancer, eg Acute Inotuzumab CD22 lymphoblastic leukemia; inotuzumabozogamicin Follicle center lymphoma; epratuzumab Non-Hodgkin lymphoma;moxetumomab Systemic lupus moxetumomab pasudotox erythematosus, hairycell leukaemia IL1 beta Acne vulgaris; gevokizumab Atherosclerosis;Behcets disease; Cardiovascular disease; Dermatomyositis; Insulindependent diabetes; Multiple myeloma; Osteoarthritis; Paraproteinemia;Polymyositis; Pyoderma gangrenosum; Scleritis; Uveitis CD20 CancerOcrelizumab Multiple sclerosis ofatumumab follicular lymphoma (eg,refractory or relapsed) diffuse large B cell lymphoma (eg, relapsed)chronic lymphocytic leukaemia (eg, first line therapy or relapsed) IL-23Crohns disease; tildrakizumab Inflammatory disease; Psoriasis BAFFAutoimmune disease Belimumab Neutrokine alpha systemic lupus Benlysta ™erythematosus Tabalumab Multiple myeloma vasculitis IL5 Asthmamepolizumab IL6 Inflammatory disease sirukumab rheumatoid arthritisLp-PLA2 Atherosclerosis darapladib diabetic macular oedema CCR9chemokine receptor Inflammatory disease Vercirnon rheumatoid arthritisCrohn's disease DOPA decarboxylase Parkinson's Disease Patrome Her2Cancer, eg, gastric cancer, Tyverb ™ EGFR breast cancer, head andTykerb ™ neck squamous cell cancer, lapatinib ADP receptorCardiovascular disease or Brilinta condition Brilique Thrombosis, eg,arterial thrombosis VEGFR Cancer, eg, Caprelsa EGFR medullary thyroidcancer LABA Respiratory disease or PT003 GFF LAMA condition, eg, COPDFactor Xa thromboembolism apixaban Oxelumab OX40 ligand AsthmaGraft-versus-host disease Tremelimumab CTLA-4 Cancer, eg, melanomaTicilimumab CD152 Autoimmune disease ipilimumab MPDL3280A PD-L1 Cancer,eg, solid tumour, MEDI4736 kidney cancer, lung cancer, melanoma, NSCLC,multiple myeloma Autoimmune disease Nivolumab PD-1 Cancer, eg, solidtumour, Keytruda kidney cancer, lung cancer, AMP-514 melanoma, NSCLC,AMP-224 multiple myeloma Autoimmune disease LIGHT TNFSF14 CD40 ligandJAK Inflammatory disease, eg, tofacitinib rheumatoid arthritis,psoriasis, chrohn's disease, IBD, ulcerative colitis, PsoriaticArthritis Nerve Growth Factor Pain tanezumab Osteoarthritis pain Her1receptor Cancer, eg, Non-Small Cell dacomitinib Her2 receptor LungCancer Her4 receptor c-MET Cancer, eg, Non-Small Cell crizotinib ALKLung Cancer Programmed cell death 1 receptor Cancer, eg, renal cellNivolumab carcinoma SLAMF7 Cancer, eg, multiple Elotuzumab CD319 myelomaCD30 Cancer, eg, Hodgkin Brentuximab lymphoma, systemic Brentuximabvedotin anaplastic large cell lymphoma, T-cell lymphoma GPR40 Diabetes,eg, diabetes Fasiglifam DPP-4 mellitus trelagliptin VEFGR-1 receptorCancer, eg, non-squamous Motesanib VEFGR-2 receptor non-small cell lungcancer Motesanib diphosphate VEFGR-3 receptor PDGFR cKit amyloid βAlzheimer's disease Solanezumab TNF alpha Inflammatory disease, eg,SIMPONI ™ rheumatoid arthritis, HUMIRA ™ psoriasis, chrohn's disease,REMICADE ™ IBD, ulcerative colitis, ENBREL ™ Psoriatic ArthritisAdalimumab IL-21 Autoimmune disease or Agonist or antagonist conditionantibody specific for Inflammatory disease or human IL-21 conditionNNC0114-0005 Rheumatoid arthritis NNC0114-0006 Crohn's disease NN8828IBD ATR-107 Ulcerative colitis Said or an anti-IL-21 Systemic lupusantibody in combination erythematosus (SLE) with an agent selected fromGraft versus host disease the group consisting of Cancer ipilimumab (eg,to treat Metastatic melanoma melanoma), an anti-PD1 Renal cell carcinomaantibody (eg, to treat solid Melanoma tumours), sunitinib (eg, to Solidtumours treat renal cell carcinoma), Acute myeloid leukaemia rituximab(eg, to treat Non- Non-Hodgkin's lymphoma Hodgkin's lymphoma), Ovariancancer sorafenib (eg, to treat renal Colorectal cancer cell carcinoma),doxorubicin (eg, to treat ovarian cancer) and cetuximab (eg, to treatcolorectal cancer).

TABLE 6 PCSK9 SEQUENCES SEQ FORM/ ID ALLELE VERSION SEQUENCE NO:AMINO ACID SEQUENCES Italics = signal sequence 1-30 Courier =pro peptide 31-152 lower case = catalytic domain 153-449 UPPER CASE =C-terminal domain 450-692 Underlined =residues changed from allele a in other sequences (aa residue numbers shown) a Pro-                                             46     53  1 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslvrlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepettlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ a Pro-QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP  2 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnlerltppryradeyqppdggsfvevylldtsiqsdhreiegrvmvtdfenvpeedgtsfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllpfaggysrvfnaacqrfaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ a Mature sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqas  3formkcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ f Pro-                                             46     53  4 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslvrlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepettlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ f Pro-QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP  5 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnlerltppryradeyqppdggsfvevylldtsiqsdhreiegrvmvtdfenvpeedgtsfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllpfaggysrvfnaacqrfaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ f, p,  Mature sipwnlerltppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqas  6aj formkcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ c Pro-                                             46     53  7 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepettlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ c Pro-QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP  8 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnlerltppryradeyqppdggsfvevylldtsiqsdhreiegrvmvtdfenvpeedgtsfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepettlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ c, q Mature sipwnlerltppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqas  9formkcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ r Pro-                                             46     53 10 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ r Pro-QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 11 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ r Mature sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqas 12formkcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ p Pro-                                             46     53 13 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLVEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ p Pro-QEDEDGDYEELVLALRSEEDGLVEAPEHGTTATFHRCAKDP 14 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ m Pro-                                             46     53 15 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvatlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ m Pro-QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 16 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvatlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ m Mature sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqas 17formkcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425               443                           474dvineawfpedqrvltpnlvatlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ e Pro-                                             46     53 18 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dviseawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ e Pro-QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 19 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dviseawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ e Mature sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqas 20formkcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425               443                           474dviseawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ h Pro-                                             46     53 21 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvatlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPPEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ h Pro-QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 22 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvatlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPPEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ h Mature sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqas 23formkcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak   425               443                           474dvineawfpedqrvltpnlvatlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPPEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ aj Pro-                                             46     53 24 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALLSEEDGLAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ aj Pro-QEDEDGDYEELVLALLSEEDGLAEAPEHGTTATFHRCAKDP 25 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ q Pro-                                             46     53 26 Form MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLVAEAPEHGTTATFHRCAKDwithPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSignalVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvSe-peedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvquenceqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ q Pro-QEDEDGDYEELVLALRSEEDGLVEAPEHGTTATFHRCAKDP 27 FormWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak    425               443                           474dvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                             619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                      670AVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ NUCLEOTIDE SEQUENCESItalics =nucleotide sequence encoding signal sequence (nucleotides 1-90)Courier = nucleotide sequence encoding pro peptide (nucleotides 91-456)lower case = nucleotide sequence encoding catalytic domain (nucleotides 457-1346) UPPER CASE =nucleotide sequence encoding C-terminal domain  (nucleotides 1347-2076)aATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC28                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg  A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC fATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC29                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg  A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC cATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC30                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC rATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC31                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC pATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC32                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGTCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC mATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC33                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccaccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC eATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC34                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                       N425S AAT to AGT atccacttctctgccaaagatgtcatcagtgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC hATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC35                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccaccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCCGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC ojATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC36                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCTTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC qATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC37                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGTCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                        N425S AAT to AGT atccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtg A443T GCC to ACCgccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC     I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    Q619P CAG to CCG E620G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA                E670G GAG to GGGCTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC

TABLE 7 Human VH3-23 Variant Alleles VH3-23 Cumulative allele haplotypefrequency SNPs a (=VH3-23*04) 0.0983 rs56069819 d 0.0087 rs56069819rs61750837 rs61752504 e 0.0046 rs56069819 rs1064090 rs1055799 j 0.0009rs56069819 rs1055799 u 0.0005 rs56069819 rs1064091 s 0.0005 rs56069819rs1064091 rs61752504 rs61750837 r 0.0005 rs56069819 rs1064090 TOTAL:0.114

TABLE 8 Exemplary anti-PCSK9 antibodies and/or antibody fragments SEQ IDNOs comprising an anti-PCSK9 monoclonal antibody or fragment thereofPatent or patent Pblication Light chain complementary determiningregions US20120020975 A1 (CDRL) SEQ ID NO: 5, 7, 9, 10, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37,38, 39, 40, 42, 44, 46, 270, 271, 272, 273, 275, 277, 286, 287, 288,297, 299, 301, 405, 407, 409, 411, 413, 415, 417, 421, 425, 429, 433,437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485; Heavychain complementary determining regions (CDRH) SEQ ID NO: 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 64, 65, 67, 69, 71, 72,74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 278, 289, 290, 291, 292,298, 300, 302, 401, 404, 406, 408, 410, 412, 414, 416, 419, 423, 427,431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483;CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120027765A1 17, 18, 19,20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,44, 46, 405, 407, 409, 411, 413, 415, 417, 465; CDRH SEQ ID NO: 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72,74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412,414, 416, 463; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, U.S. Pat.No. 8,168,762B2 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417,465; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60,62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89,91, 404, 406, 408, 410, 412, 414, 416, 463; CDRL SEQ ID NO: 5, 7, 9, 10,12, 13, 15, 16, US20120020976A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 222, 229, 238, 405,407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78,79, 80, 81, 83, 85, 87, 89, 91, 247, 256, 265, 404, 406, 408, 410, 412,414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130085265A117, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38,39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485;CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62,64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91,404, 406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7,9, 10, 12, 13, 15, 16, US20130079501A1 17, 18, 19, 20, 21, 22, 23, 24,26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407,409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76,77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414,416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16,US20120213797A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417,158, 162, 395, 473, 477; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80,81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 180, 175,308, 368, 471, 475; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16,US20120251544A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417;CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62,64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91,404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13,15, 16, US20130052201A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31,32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413,415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80,81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463,483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130058944A1 17,18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39,40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRHSEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64,65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404,406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9,10, 12, 13, 15, 16, US20130079502A1 17, 18, 19, 20, 21, 22, 23, 24, 26,28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409,411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81,83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO:5, 7, 9, 10, 12, 13, 15, 16, US20130245235A1 17, 18, 19, 20, 21, 22, 23,24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405,407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78,79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416;

TABLE 9 Human variable & constant variants distributed over severalhuman ethnic populations-useful for ligand tailoring Nucleotide AminoVariation² Hom Human Acid (NCBI Freq⁷ Gene Example Position dbSNPVariant (Het + Segment Human & reference Nucleotide Human No. Het HomCum Type Allele¹ Variation number)³ Position Populations⁴ Individs⁵Freq⁶ freq⁸) Freq⁹ IGHG1 IGHG1*01 204D GAT 14:106208086 A 153 0.4000.096 0.296 (CH3 (forward strand) (European (0.496) variation) ancestry)IGHG1*03 204E GAG 14:106208086 A 366 0.400 0.504 0.704 (CH3 (rs1045853)(forward strand) (European (0.904) variation) ancestry IGHG1*01 206L CTG14:106208082 A 0.358 0.104 0.283 (CH3 (forward strand) (European (0.462)variation) ancestry) IGHG1*03 206M ATG 14:106208082 A 0.358 0.538 0.717(CH3 (rs11621259) (forward strand) (European (0.896) variation)ancestry) IGHG2 IGHG2*01 72P CCC 14:106110914 B 0.336 0.540 0.708 (CH1(forward strand) (0.876) variation) IGHG2*02 72T ACC 14:106110914 B0.336 0.124 0.292 (CH1 (rs11627594) (forward strand) variation) IGHG2*0175N AAC 14:106110904 A 0.007 0.993 0.997 (CH1 (forward strand)variation) IGHG2*04 75S AGC 14:106110904 A 0.007 0.004 (CH1(rs201590297) (forward strand) variation) IGHG2*01 76F TTC (CH1variation) IGHG2*04 76L TTG (CH1 variation) IGHG2*01 161V GTG14:10611013 B 0.342 0.539 0.711 (CH2 (forward strand) (0.881) variation)IGHG2*02 161M ATG 14:10611013 B 0.342 0.118 0.289 (CH2 (rs8009156)(forward strand) (0.46) variation) IGHG2*01 257A GCC 14:106109752 C0.199 0.493 0.592 (CH3 (forward strand) (0.692) variation) D 0.007 0.9920.995 (0.999) IGHG2*06 257S TCC 14:106109752 C 0.199 0.308 0.408 (CH3(rs4983499) (forward strand) (0.507) variation) D 0.007 0.002 0.005(0.009) ¹IMGT notation (ww.imgt.org); refer to figures for other allelescomprising this variation. ²SNP Underlined in Codon. ³NCBI dbSNP Build138 released on Apr. 25, 2013. ⁴Human population used for representativevariant frequency analysis. Populations A This populationincluded 662participants of European descent from the ClinSeq project, all of whomhad undergone whole-exome sequencing using Agilent's 38 Mb or 50 Mbcapture kit. B 1000 Genomes database. C ESP6500: African_American. DESP6500: European_American. ⁵Number of individuals in representativepopulation found to have the allele. ⁶Heterozygous human genotypefrequency, ie, cumulative frequency of all genotypes having oneoccurrence of the variant allele and one occurrence of another allele(heterozygous state), eg, ac genotype in the population. ⁷Homozygoushuman genotype frequency, ie, cumulative frequency of two occurrences ofthe variant allele (homozygous state), eg, cc genotype in thepopulation. ⁸Total human genotype frequency, ie, total of heterozygousplus homozygous human genotype frequencies. ⁹Cumulative human allelefrequency of all occurrences of the variant allele in the population.

TABLE 10 FURTHER SEQUENCES SEQ ID Human NO: AlleleNucleotide/Amino Acid Sequence HEAVY CHAIN ALLELES 41 IGHG1*01gcctccaccaagggcccatcggtcttccccctggcaccctcctccaaga (CH1 + Hinge +gcacctctggg CH2 + CH3 +ggcacagcggccctgggctgcctggtcaaggactacttccccgaaccgg CH-S) tgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcc tacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgg gcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaaga aagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaac tcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct cccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtca agttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggc tgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgaga aaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccat cccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatc ccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcaca accactacacgcagaagagcctctccctgtctccgggtaaa 42ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSR D E LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK D  = position 204 L  = position 206 43IGHG2*01 gcctccaccaagggcccatcggtcttccccctggcgccctgctccagga (CH1 +Hinge + gcacctccgag CH2 + CH3 +agcacagccgccctgggctgcctggtcaaggactacttccccgaaccgg CH-S) tgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcc tacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcg gcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaaga cagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggac cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggt acgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaaca gcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaagg agtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctcca aaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggaga tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgc tggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc agcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc agaagagcctctccctgtctccgggtaaa 44 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTV P SS NF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG V EVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDI A VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK P  = position 72 N  = position 75F  = position 76 V  = position 161 A  = position 257 45 IGHV1-18*01caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcct cagtgaaggtctcctgcaaggcttctggttacacctttaccagctatggtatcagctggg tgcgacaggcccctggacaagggcttgagtggatgggatggatcagcgcttacaatggta acacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccacga gcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtg cgagaga 46QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCAR 47IGHV1-46*01 caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtt tcctgcaaggcatctggatacaccttcaccagctactatatgcactgggtgcgacaggcc cctggacaagggcttgagtggatgggaataatcaaccctagtggtggtagcacaagctac gcacagaagttccagggcagagtcaccatgaccagggacacgtccacgagcacagtctac atggagctgagcagcctgagatctgaggacacggccgtgtattactgtg cgagaga 48QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARLIGHT CHAIN ALLELES 49 IGKC*01cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtca cagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaag cagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgc ccgtcacaaagagcttcaacaggggagagtgt 50RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK V YA C EVTHQGLSSPVTKSFNRGEC V  = position 84 C = position 87 51 IGLC2*01ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctg aggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggag ccgtgacagtggcttggaaagcagatagcagccccgtcaaggcgggagtggagaccacca caccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctg agcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtgg agaagacagtggcccctacagaatgttca 52 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSK QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 53 IGKV4-1*01atggtgttgcagacccaggtcttcatttctctgttgctctggatctctg gtgcctacggggacatcgtgatgacccagtctccagactccctggctgtgtctctgggcg agagggccaccatcaactgcaagtccagccagagtgttttatacagctccaacaataaga actacttagcttggtaccagcagaaaccaggacagcctcctaagctgctcatttactggg catctacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatt tcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaat attatagtact cctcc 54DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYST P 55IGKV1-13*02 atggacatgagggtccccgctcagctcctggggcttctgctgctctggctcccagcaggt gccagatgtgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagac agagtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcctggtatcag cagaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggg gtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttaccctcagtgc cagatgtgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacag agtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcctggtatcagca gaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggt cccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcct gcagcctgaagattttgcaacttattactgtcaacagtttaatagttac cctca 57IGKJ2*01 tgtacacttttggccaggggaccaagctggagatcaaac 58 YTFGQGTKLEIK 59IGLJ2*01 tgtggtattcggcggagggaccaagctgaccgtcctag 60 VVFGGGTKLTVL 61An IGHG1*01 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGHeavy Chain VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV ConstantEPKSCDKTHTCPPCPAPELLGGPS RegionVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 62 An IGKC*01RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS Kappa LightGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV Chain TKSFNRGECConstant Region 63 An IGHG2*01ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Heavy ChainVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ConstantERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH RegionEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 64 An IGLC2*01QPKAAPSVTLEPPSSEELQANKATLVCLISDFYPGAVIVAWKADSSPVK LambdaAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT Light Chain VAPTECSConstant Region 65 An IGHG2*01ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Heavy ChainVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ConstantERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH RegionEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 66 An IGKC*01RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS Kappa LightGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV Chain TKSFNRGECConstant Region

1. A method of treating or preventing diabetic macular edema (DME) orbranch retinal vein occlusion (BRVO) in a human, the method comprisingadministering to said human an antibody, antibody fragment, or a trapthat specifically binds to a human VEGF-A; wherein the antibody,antibody fragment, or trap comprises a human gamma-1 heavy chainconstant region that comprises an Asp corresponding to position 204 ofSEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42and wherein (i) said human comprises and expresses an IGHG1*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-1 heavy chain constant regionscomprising an Asp corresponding to position 204 of SEQ ID NO: 42 or aLeu corresponding to position 206 of SEQ ID NO: 42 and (ii) said humancomprises a nucleotide sequence encoding said VEGF-A.
 2. The method ofclaim 1, wherein the antibody, antibody fragment, or trap comprises ahuman gamma-1 heavy chain constant region that comprises an Aspcorresponding to position 204 of SEQ ID NO: 42 and a Leu correspondingto position 206 of SEQ ID NO:
 42. 3. The method of claim 1, wherein theantibody, antibody fragment, or trap comprises an IGHG1*01 human heavychain constant region.
 4. The method of claim 1, further comprising astep of selecting a human comprising said nucleotide sequence of (ii)before administering to said human the antibody, antibody fragment, ortrap.
 5. The method of claim 1, wherein the antibody, antibody fragment,or trap is administered by intravenous or subcutaneous administrationand/or is comprised in an injectable preparation.
 6. The method of claim1, wherein the antibody or antibody fragment is a human antibody orhuman antibody fragment.